CSAPK-2 protein and uses therefor

ABSTRACT

The present invention provides a novel protein kinase, CSAPK-2, as well as CSAPK-2 fusion proteins, antigenic peptides and anti-CSAPK-2 antibodies.

RELATED APPLICATIONS

This application is a divisional application of Ser. No. 09/163,115filed on Sep. 29, 1998, pending. The contents of all of theaforementioned application(s) are incorporated herein by reference,which claims priority to U.S. provisional Application No. 60/099,657,filed on Sep. 9, 1998, incorporated herein in its entirety by thisreference.

BACKGROUND OF THE INVENTION

Phosphate tightly associated with protein has been known since the latenineteenth century. Since then, a variety of covalent linkages ofphosphate to proteins have been found. The most common involveesterification of phosphate to serine, threonine, and tyrosine withsmaller amounts being linked to lysine, arginine, histidine, asparticacid, glutamic acid, and cysteine. The occurrence of phosphorylatedproteins implies the existence of one or more protein kinases capable ofphosphorylating amino acid residues on proteins, and also of proteinphosphatases capable of hydrolyzing phosphorylated amino acid residueson proteins.

Protein kinases play critical roles in the regulation of biochemical andmorphological changes associated with cellular growth and division(D'Urso, G. et al. (1990) Science 250: 786-791; Birchmeier. C. et al.(1993) Bioessays 15: 185-189). They serve as growth factor receptors andsignal transducers and have been implicated in cellular transformationand malignancy (Hunter, T. et al. (1992) Cell 70: 375-387; Posada, J. etal. (1992) Mol. Biol. Cell 3: 583-592; Hunter, T. et al. (1994) Cell 79:573-582). For example, protein kinases have been shown to participate inthe transmission of signals from growth-factor receptors (Sturgill, T.W. et al. (1988) Nature 344: 715-718; Gomez, N. et al. (1991) Nature353: 170-173), control of entry of cells into mitosis (Nurse, P. (1990)Nature 344: 503-508; Maller, J. L. (1991) Curr. Opin. Cell Biol. 3:269-275) and regulation of actin bundling (Husain-Chishti, A. et al.(1988) Nature 334: 718-721). Protein kinases can be divided into twomain groups based on either amino acid sequence similarity orspecificity for either serine/threonine or tyrosine residues. A smallnumber of dual-specificity kinases are structurally like theserine/threonine-specific group. Within the broad classification,kinases can be further sub-divided into families whose members share ahigher degree of catalytic domain amino acid sequence identity and alsohave similar biochemical properties. Most protein kinase family membersalso share structural features outside the kinase domain that reflecttheir particular cellular roles. These include regulatory domains thatcontrol kinase activity or interaction with other proteins (Hanks, S. K.et al. (1988) Science 241: 42-52).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel nucleic acid molecules and proteins encoded by such nucleic acidmolecules, referred to herein as "Cardiovascular System AssociatedProtein Kinase" ("CSAPK") proteins. The CSAPK nucleic acid and proteinmolecules of the present invention are useful as modulating agents inregulating a variety of cellular processes, e.g., cardiac cellularprocesses. Accordingly, in one aspect, this invention provides isolatednucleic acid molecules encoding CSAPK proteins or biologically activeportions thereof, as well as nucleic acid fragments suitable as primersor hybridization probes for the detection of CSAPK-encoding nucleicacids.

In one embodiment, a CSAPK nucleic acid molecule of the invention is atleast 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or morehomologous to a nucleotide sequence (e.g., to the entire length of thenucleotide sequence) including SEQ ID NO:1, SEQ ID NO:3, or a complementthereof. In another embodiment, a CSAPK nucleic acid molecule is 50%,54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98% homologous toa nucleotide sequence including SEQ ID NO:4, SEQ ID NO:6, or acomplement thereof. In yet another embodiment, a CSAPK nucleic acidmolecule is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% homologousto a nucleotide sequence including SEQ ID NO:7, SEQ ID NO:9, or acomplement thereof. In yet another embodiment, a CSAPK nucleic acidmolecule is 60%, 65%, 70%, 73%, 75%, 80%, 85%, 86%, 90%, 95%, 98%homologous to a nucleotide sequence including SEQ ID NO:10, SEQ IDNO:12, or a complement thereof. In a further embodiment, a CSAPK nucleicacid molecule is 60%, 65%, 70%, 75%, 78%, 80%, 85%, 90%, 95%, 98%homologous to a nucleotide sequence including SEQ ID NO:13, SEQ IDNO:15, or a complement thereof.

In a preferred embodiment, the isolated nucleic acid molecule includesthe nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof.In another embodiment, the nucleic acid molecule includes SEQ ID NO:3and nucleotides 1-296 of SEQ ID NO:1. In yet another embodiment, thenucleic acid molecule includes SEQ ID NO:3 and nucleotides 1202-4137 ofSEQ ID NO:1. In another preferred embodiment, the nucleic acid moleculehas the nucleotide sequence shown in SEQ ID NO:1 or 3. In anotherpreferred embodiment, the nucleic acid molecule comprises a fragment ofat least 509 nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQID NO:3, or a complement thereof.

In another preferred embodiment, the isolated nucleic acid moleculeincludes the nucleotide sequence shown SEQ ID NO:4 or 6, or a complementthereof. In another embodiment, the nucleic acid molecule includes SEQID NO:6 and nucleotides 1-46 of SEQ ID NO:4. In yet another embodiment,the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 1411-2120of SEQ ID NO:4. In another preferred embodiment, the nucleic acidmolecule has the nucleotide sequence shown in SEQ ID NO:4 or 6.

In another preferred embodiment, the isolated nucleic acid moleculeincludes the nucleotide sequence shown SEQ ID NO:7 or 9, or a complementthereof. In another embodiment, the nucleic acid molecule includes SEQID NO:9 and nucleotides 1-50 of SEQ ID NO:7. In yet another embodiment,the nucleic acid molecule includes SEQ ID NO:9 and nucleotides 1793-2454of SEQ ID NO:7. In another preferred embodiment, the nucleic acidmolecule has the nucleotide sequence shown in SEQ ID NO:7 or 9.

In another preferred embodiment, the isolated nucleic acid moleculeincludes the nucleotide sequence shown SEQ ID NO:10 or 12, or acomplement thereof. In another embodiment, the nucleic acid moleculeincludes SEQ ID NO:12 and nucleotides 1-274 of SEQ ID NO:10. In yetanother embodiment, the nucleic acid molecule includes SEQ ID NO:12 andnucleotides 755-1864 of SEQ ID NO:10. In another preferred embodiment,the nucleic acid molecule has the nucleotide sequence shown in SEQ IDNO:10 or 12.

In another preferred embodiment, the isolated nucleic acid moleculeincludes the nucleotide sequence shown in SEQ ID NO:13 or 15, or acomplement thereof. In yet another preferred embodiment, the nucleicacid molecule has the nucleotide sequence shown in SEQ ID NO:13 or 15.

In another embodiment, a CSAPK nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequencesufficiently homologous to the amino acid sequence of SEQ ID NO:2, SEQID NO:5, SEQ ID 8, SEQ ID NO:11, or SEQ ID NO:14. In a preferredembodiment, a CSAPK nucleic acid molecule includes a nucleotide sequenceencoding a protein having an amino acid sequence at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 81%, 85%, 90%, 95%, 98% or more homologous to anamino acid sequence including SEQ ID NO:2 (e.g., the entire amino acidsequence of SEQ ID NO:2). In another preferred embodiment, a CSAPKnucleic acid molecule includes a nucleotide sequence encoding a proteinhaving an amino acid sequence at least 42%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to an amino acidsequence including SEQ ID NO:5 (e.g., the entire amino acid sequence ofSEQ ID NO:5). In yet another preferred embodiment, a CSAPK nucleic acidmolecule includes a nucleotide sequence encoding a protein having anamino acid sequence at least 41%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98% or more homologous to an amino acid sequenceincluding SEQ ID NO:8 (e.g., the entire amino acid sequence of SEQ IDNO:8). In a further preferred embodiment, a CSAPK nucleic acid moleculeincludes a nucleotide sequence encoding a protein having an amino acidsequence at least 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% ormore homologous to an amino acid sequence including SEQ ID NO:11 (e.g.,the entire amino acid sequence of SEQ ID NO:11). In another preferredembodiment, a CSAPK nucleic acid molecule includes a nucleotide sequenceencoding a protein having an amino acid sequence at least 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or more homologous to an amino acidsequence including SEQ ID NO:14 (e.g., the entire amino acid sequence ofSEQ ID NO:14).

In another preferred embodiment, an isolated nucleic acid moleculeencodes the amino acid sequence of a human CSAPK. In yet anotherpreferred embodiment, the nucleic acid molecule includes a nucleotidesequence encoding a protein which includes the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:5, SEQ ID 8, SEQ ID NO:11, or SEQ ID NO:14. Inyet another preferred embodiment, the nucleic acid molecule includes anucleotide sequence encoding a protein having the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:5, SEQ ID 8, SEQ ID NO:11, or SEQ ID NO:14.

Another embodiment of the invention features nucleic acid moleculespreferably CSAPK nucleic acid molecules, which specifically detect CSAPKnucleic acid molecules relative to nucleic acid molecules encodingnon-CSAPK proteins. For example, in one embodiment, such a nucleic acidmolecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, or 800 nucleotides in length and hybridizesunder stringent conditions to a nucleic acid molecule comprising thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID 7, SEQ IDNO:10, SEQ ID NO:13, or a complement thereof.

In other preferred embodiments, the nucleic acid molecule encodes anaturally occurring allelic variant of a polypeptide which includes theamino acid sequence of SEQ ID NO:2, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule which includes SEQ ID NO:1 or SEQID NO:3 under stringent conditions. In other preferred embodiments, thenucleic acid molecule encodes a naturally occurring allelic variant of apolypeptide which includes the amino acid sequence of SEQ ID NO:5,wherein the nucleic acid molecule hybridizes to a nucleic acid moleculewhich includes SEQ ID NO:4 or SEQ ID NO:6 under stringent conditions. Inother preferred embodiments, the nucleic acid molecule encodes anaturally occurring allelic variant of a polypeptide which includes theamino acid sequence of SEQ ID NO:8, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule which includes SEQ ID NO:7 or SEQID NO:9 under stringent conditions. In other preferred embodiments, thenucleic acid molecule encodes a naturally occurring allelic variant of apolypeptide which includes the amino acid sequence of SEQ ID NO:11,wherein the nucleic acid molecule hybridizes to a nucleic acid moleculewhich includes SEQ ID NO:10 or SEQ ID NO:12 under stringent conditions.In other preferred embodiments, the nucleic acid molecule encodes anaturally occurring allelic variant of a polypeptide which includes theamino acid sequence of SEQ ID NO:14, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule which includes SEQ ID NO:13 or SEQID NO:15 under stringent conditions.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a CSAPK nucleic acid molecule, e.g., thecoding strand of a CSAPK nucleic acid molecule.

Another aspect of the invention provides a vector comprising a CSAPKnucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. The inventionalso provides a method for producing a protein, preferably a CSAPKprotein, by culturing in a suitable medium, a host cell, e.g., amammalian host cell such as a non-human mammalian cell, of the inventioncontaining a recombinant expression vector, such that the protein isproduced.

Another aspect of this invention features isolated or recombinant CSAPKproteins and polypeptides. In one embodiment, the isolated protein,preferably a CSAPK-1 protein, includes at least one Ser/Thr kinasedomain, and at least one ATP-binding site. In another embodiment, theisolated protein, preferably a CSAPK-1 protein, includes at least oneSer/Thr kinase domain, and at least one ATP-binding site and has anamino acid sequence which is at least 51%, 55%, 60%, 65%, 70%, 75%, 80%,81%, 85%, 90%, 95%, 99% or more homologous to an amino acid sequenceincluding SEQ ID NO:2. In yet another embodiment, the isolated protein,preferably a CSAPK-1 protein, includes at least one Ser/Thr kinasedomain, and at least one ATP-binding site and is expressed and/orfunctions in cells of the cardiovascular system. In an even furtherembodiment, the isolated protein, preferably a CSAPK-1 protein, includesat least one Ser/Thr kinase domain, and at least one ATP-binding siteand plays a role in signalling pathways associated with cellular growth,e.g., signalling pathways associated with cell cycle regulation. Inanother embodiment, the isolated protein, preferably a CSAPK-1 protein,includes at least one Ser/Thr kinase domain, and at least oneATP-binding site and is encoded by a nucleic acid molecule having anucleotide sequence which hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1 or SEQ ID NO:3.

In another embodiment, the isolated protein, preferably a CSAPK-2protein, includes at least one dual specificity kinase catalytic domainand at least one ATP-binding site. In another embodiment, the isolatedprotein, preferably a CSAPK-2 protein, includes at least one dualspecificity kinase catalytic domain, at least one leucine zipper-basicregion, and at least one ATP-binding site. In another embodiment, theisolated protein, preferably a CSAPK-2 protein, includes at least onedual specificity kinase catalytic domain and at least one ATP-bindingsite and has an amino acid sequence which is at least 42%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous toan amino acid sequence including SEQ ID NO:5. In yet another embodiment,the isolated protein, preferably a CSAPK-2 protein, includes at leastone dual specificity kinase catalytic domain, and at least oneATP-binding site and is expressed and/or functions in cells of thecardiovascular system. In an even further embodiment, the isolatedprotein, preferably a CSAPK-2 protein, includes at least one dualspecificity kinase catalytic domain, and at least one ATP-binding siteand plays a role in signalling pathways associated with cellular growth,e.g., signalling pathways associated with cell cycle regulation. Inanother embodiment, the isolated protein, preferably a CSAPK-2 protein,includes at least one dual specificity kinase catalytic domain, and atleast one ATP-binding site and is encoded by a nucleic acid moleculehaving a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:4 or SEQ ID NO:6.

In yet another embodiment, the isolated protein, preferably a CSAPK-3protein, includes at least one Ser/Thr kinase domain, and at least oneATP-binding site. In another embodiment, the isolated protein,preferably a CSAPK-3 protein, includes at least one Ser/Thr kinasedomain, and at least one ATP-binding site and has an amino acid sequencewhich is at least 41%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98% or more homologous to an amino acid sequence including SEQ IDNO:8. In yet another embodiment, the isolated protein, preferably aCSAPK-3 protein, includes at least one Ser/Thr kinase domain, and atleast one ATP-binding site and is expressed and/or functions in cells ofthe cardiovascular system. In an even ftirther embodiment, the isolatedprotein, preferably a CSAPK-3 protein, includes at least one Ser/Thrkinase domain, and at least one ATP-binding site and plays a role insignalling pathways associated with cellular growth, e.g., signallingpathways associated with cell cycle regulation. In another embodiment,the isolated protein, preferably a CSAPK-3 protein, includes at leastone Ser/Thr kinase domain, and at least one ATP-binding site and isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:7 or SEQ IDNO:9.

In another embodiment, the isolated protein, preferably a CSAPK-4protein, includes at least one Ser/Thr kinase domain, and at least oneATP-binding site. In another embodiment, the isolated protein,preferably a CSAPK-4 protein, includes at least one Ser/Thr kinasedomain, and at least one ATP-binding site and has an amino acid sequencewhich is at least 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% ormore homologous to an amino acid sequence including SEQ ID NO:11. In yetanother embodiment, the isolated protein, preferably a CSAPK-4 protein,includes at least one Ser/Thr kinase domain, and at least oneATP-binding site and is expressed and/or functions in cells of thecardiovascular system. In an even further embodiment, the isolatedprotein, preferably a CSAPK-4 protein, includes at least one Ser/Thrkinase domain, and at least one ATP-binding site and plays a role insignalling pathways associated with cellular growth, e.g., signallingpathways associated with cell cycle regulation. In another embodiment,the isolated protein, preferably a CSAPK-4 protein, includes at leastone Ser/Thr kinase domain, and at least one ATP-binding site and isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:10 or SEQ IDNO:12.

In another embodiment, the isolated protein, preferably a human CSAPK-5protein, includes at least one Ser/Thr kinase domain, and at least oneATP-binding site. In yet another embodiment, the isolated protein,preferably a human CSAPK-5 protein, includes at least one Ser/Thr kinasedomain, and at least one ATP-binding site and has an amino acid sequencewhich is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or morehomologous to an amino acid sequence including SEQ ID NO:14. In yetanother embodiment, the isolated protein, preferably a human CSAPK-5protein, includes at least one Ser/Thr kinase domain, and at least oneATP-binding site and is expressed and/or functions in cells of thecardiovascular system. In an even further embodiment, the isolatedprotein, preferably a human CSAPK-5 protein, includes at least oneSer/Thr kinase domain, and at least one ATP-binding site and plays arole in signalling pathways associated with cellular growth, e.g.,signalling pathways associated with cell cycle regulation. In anotherembodiment, the isolated protein, preferably a human CSAPK-5 protein,includes at least one Ser/Thr kinase domain, and at least oneATP-binding site and is able to interact with Nck (described in Lehmanet al. (1990) Nucleic Acids Res. 18:1048). In another embodiment, theisolated protein, preferably a human CSAPK-5 protein, includes at leastone Ser/Thr kinase domain, and at least one ATP-binding site and isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:13 or SEQ IDNO:15.

In another embodiment, the isolated protein, preferably a CSAPK protein,has an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID 8, SEQ ID NO:11, or SEQ IDNO:14. In a preferred embodiment, the protein, preferably a CSAPKprotein, has an amino acid sequence at least about 41%, 42%, 45%, 50%,55%, 59%, 60%, 65%, 70%, 75%, 80%, 81%, 85%, 90%, 95%, 98% or morehomologous to an amino acid sequence including SEQ ID NO:2, SEQ ID NO:5,SEQ ID 8, SEQ ID NO:11, or SEQ ID NO:14 (e.g., the entire amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID 8, SEQ ID NO:11, or SEQ IDNO:14). In another embodiment, the invention features fragments of theproteins having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQID 8, SEQ ID NO:11, or SEQ ID NO:14, wherein the fragment comprises atleast 15 amino acids (e.g., contiguous amino acids) of the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID 8, SEQ ID NO:11, or SEQ IDNO:14, respectively. In another embodiment, the protein, preferably aCSAPK protein, has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5,SEQ ID 8, SEQ ID NO:11, or SEQ ID NO:14.

Another embodiment of the invention features an isolated protein,preferably a CSAPK protein, which is encoded by a nucleic acid moleculehaving a nucleotide sequence at least about 50%, 54%, 55%, 60%, 62%,65%, 70%, 75%, 78%, 80%, 85%, 90%, 95%, 97%, 98% or more homologous to anucleotide sequence (e.g., to the entire length of the nucleotidesequence) including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, orSEQ ID NO:15, or a complement thereof. This invention further featuresan isolated protein, preferably a CSAPK protein, which is encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, or SEQ ID NO:15, or a complement thereof.

The proteins of the present invention or biologically active portionsthereof, can be operatively linked to a non-CSAPK polypeptide (e.g.,heterologous amino acid sequences) to form fusion proteins. Theinvention further features antibodies, such as monoclonal or polyclonalantibodies, that specifically bind proteins of the invention, preferablyCSAPK proteins. In addition, the CSAPK proteins or biologically activeportions thereof can be incorporated into pharmaceutical compositions,which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of a CSAPK nucleic acid molecule, protein or polypeptide ina biological sample by contacting the biological sample with an agentcapable of detecting a CSAPK nucleic acid molecule, protein orpolypeptide such that the presence of a CSAPK nucleic acid molecule,protein or polypeptide is detected in the biological sample.

In another aspect, the present invention provides a method for detectingthe presence of CSAPK activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofCSAPK activity such that the presence of CSAPK activity is detected inthe biological sample.

In another aspect, the invention provides a method for modulating CSAPKactivity comprising contacting a cell capable of expressing CSAPK withan agent that modulates CSAPK activity such that CSAPK activity in thecell is modulated. In one embodiment, the agent inhibits CSAPK activity.In another embodiment, the agent stimulates CSAPK activity. In oneembodiment, the agent is an antibody that specifically binds to a CSAPKprotein. In another embodiment, the agent modulates expression of CSAPKby modulating transcription of a CSAPK gene or translation of a CSAPKmRNA. In yet another embodiment, the agent is a nucleic acid moleculehaving a nucleotide sequence that is antisense to the coding strand of aCSAPK mRNA or a CSAPK gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant CSAPKprotein or nucleic acid expression or activity by administering an agentwhich is a CSAPK modulator to the subject. In one embodiment, the CSAPKmodulator is a CSAPK protein. In another embodiment the CSAPK modulatoris a CSAPK nucleic acid molecule. In yet another embodiment, the CSAPKmodulator is a peptide, peptidomimetic, or other small molecule. In apreferred embodiment, the disorder characterized by aberrant CSAPKprotein or nucleic acid expression is a cellular growth relateddisorder, e.g., a cardiovascular disorder.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aCSAPK protein; (ii) mis-regulation of the gene; and (iii) aberrantpost-translational modification of a CSAPK protein, wherein a wild-typeform of the gene encodes a protein with a CSAPK activity.

In another aspect the invention provides a method for identifying acompound that binds to or modulates the activity of a CSAPK protein, byproviding an indicator composition comprising a CSAPK protein havingCSAPK activity, contacting the indicator composition with a testcompound, and determining the effect of the test compound on CSAPKactivity in the indicator composition to identify a compound thatmodulates the activity of a CSAPK protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DERSCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cDNA sequence and predicted amino acid sequence ofhuman CSAPK-1. The nucleotide sequence corresponds to nucleic acids 1 to4137 of SEQ ID NO:1. The amino acid sequence corresponds to amino acids1 to 302 of SEQ ID NO:2. The coding region without the 5' and 3'untranslated regions of the human CSAPK-1 gene is shown in SEQ ID NO:3.

FIG. 2 depicts the cDNA sequence and predicted amino acid sequence ofhuman CSAPK-2. The nucleotide sequence corresponds to nucleic acids 1 to2120 of SEQ ID NO:4. The amino acid sequence corresponds to amino acids1 to 455 of SEQ ID NO:5. The coding region without the 5' and 3'untranslated regions of the human CSAPK-2 gene is shown in SEQ ID NO:6.

FIG. 3 depicts the cDNA sequence and predicted amino acid sequence ofhuman CSAPK-3. The nucleotide sequence corresponds to nucleic acids 1 to2454 of SEQ ID NO:7. The amino acid sequence corresponds to amino acids1 to 581 of SEQ ID NO:8. The coding region without the 5' and 3'untranslated regions of the human CSAPK-3 gene is shown in SEQ ID NO:9.

FIG. 4 depicts the cDNA sequence and predicted amino acid sequence ofhuman CSAPK-4. The nucleotide sequence corresponds to nucleic acids 1 to1864 of SEQ ID NO:10. The amino acid sequence corresponds to amino acids1 to 160 of SEQ ID NO:11.

The coding region without the 5' and 3' untranslated regions of thehuman CSAPK-4 gene is shown in SEQ ID NO:12.

FIG. 5 depicts the cDNA sequence and predicted amino acid sequence ofhuman CSAPK-5. The nucleotide sequence corresponds to nucleic acids 1 to1333 of SEQ ID NO:13. The amino acid sequence corresponds to amino acids1 to 444 of SEQ ID NO:14. The coding region without the 5' and 3'untranslated regions of the human CSAPK-5 gene is shown in SEQ ID NO:15.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as "Cardiovascular System AssociatedProtein Kinase" or "CSAPK" nucleic acid and polypeptide molecules, whichplay a role in or function in signalling pathways associated withcellular growth. In one embodiment, the CSAPK molecules modulate theactivity of one or more proteins involved in cellular growth ordifferentiation, e.g., cardiac cell growth or differentiation. Inanother embodiment, the CSAPK molecules of the present invention arecapable of modulating the phosphorylation state of a CSAPK molecule orone or more proteins involved in cellular growth or differentiation,e.g., cardiac cell growth or differentiation.

In a preferred embodiment, the CSAPK molecules are protein kinases whichare expressed and/or function in cells of the cardiovascular system,e.g., cells of the heart, the blood vessels, and/or the blood.

As used herein, the term "protein kinase" includes a protein orpolypeptide which is capable of modulating its own phosphorylation stateor the phosphorylation state of another protein or polypeptide. Proteinkinases can have a specificity for (i.e., a specificity tophosphorylate) serine/threonine residues, tyrosine residues, or bothserine/threonine and tyrosine residues, e.g., the dual specificitykinases. As referred to herein, protein kinases preferably include acatalytic domain of about 200-400 amino acid residues in length,preferably about 200-300 amino acid residues in length, or morepreferably about 250-300 amino acid residues in length, which includespreferably 5-20, more preferably 5-15, or preferably 11 highly conservedmotifs or subdomains separated by sequences of amino acids with reducedor minimal conservation. Specificity of a protein kinase forphosphorylation of either tyrosine or serine/threonine can be predictedby the sequence of two of the subdomains (VIb and VIII) in whichdifferent residues are conserved in each class (as described in, forexample, Hanks et al. (1988) Science 241:42-52) the contents of whichare incorporated herein by reference). These subdomains are alsodescribed in further detail herein.

Protein kinases play a role in signalling pathways associated withcellular growth. For example, protein kinases are involved in theregulation of signal transmission from cellular receptors, e.g.,growth-factor receptors; entry of cells into mitosis; and the regulationof cytoskeleton function, e.g., actin bundling. Thus, the CSAPKmolecules of the present invention may be involved in: 1) the regulationof transmission of signals from cellular receptors, e.g., cardiac cellgrowth factor receptors; 2) the modulation of the entry of cells, e.g.,cardiac precursor cells, into mitosis; 3) the modulation of cellulardifferentiation; 4) the modulation of cell death; and 5) the regulationof cytoskeleton function, e.g., actin bundling.

Inhibition or over stimulation of the activity of protein kinasesinvolved in signaling pathways associated with cellular growth can leadto perturbed cellular growth, which can in turn lead to cellular growthrelated disorders. As used herein, a "cellular growth related disorder"includes a disorder, disease, or condition characterized by aderegulation, e.g., an upregulation or a downregulation, of cellulargrowth. Cellular growth deregulation may be due to a deregulation ofcellular proliferation, cell cycle progression, cellular differentiationand/or cellular hypertrophy. Examples of cellular growth relateddisorders include cardiovascular disorders such as heart failure,hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathiccardiomyopathy, or angina; proliferative disorders or differentiativedisorders such as cancer, e.g., melanoma, prostate cancer, cervicalcancer, breast cancer, colon cancer, or sarcoma.

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as CSAPK protein and nucleic acidmolecules, which comprise a family of molecules having certain conservedstructural and functional features. The term "family" when referring tothe protein and nucleic acid molecules of the invention is intended tomean two or more proteins or nucleic acid molecules having a commonstructural domain or motif and having sufficient amino acid ornucleotide sequence homology as defined herein. Such family members canbe naturally or non-naturally occurring and can be from either the sameor different species. For example, a family can contain a first proteinof human origin, as well as other, distinct proteins of human origin oralternatively, can contain homologues of non-human origin. Members of afamily may also have common functional characteristics.

One embodiment of the invention features CSAPK nucleic acid molecules,preferably human CSAPK molecules, e.g., CSAPK-1, CSAPK-2, CSAPK-3,CSAPK-4, and CSAPK-5, which were identified from cDNA libraries madefrom hearts of patients with congestive heart failure (CHF) of ischemicand idiopathic origin. The CSAPK nucleic acid and protein molecules ofthe invention are described in further detail in the followingsubsections.

A. The CSAPK-1 Nucleic Acid and Protein Molecules

In one embodiment, the isolated proteins of the present invention,preferably CSAPK-1 proteins, are identified based on the presence of atleast one "Ser/Thr kinase site" and at least one "ATP-binding region."As used herein, the term "Ser/Thr kinase site" includes an amino acidsequence of about 200-400 amino acid residues in length, preferably200-300 amino acid residues in length, and more preferably 250-300 aminoacid residues in length, which is conserved in kinases whichphosphorylate serine and threonine residues and found in the catalyticdomain of Ser/Thr kinases. Preferably, the Ser/Thr kinase site includesthe following amino acid consensus sequence X₉ -g-X-G-X₄ -V-X₁₂-K-X-.sub.(10-19) -E-X₆₆ -h-X₈ -h-r-D-X-K-X₂ -N-X₁₇ -K-X₂ -D-f-g-X₂₁-p-X₁₃ -w-X₃ -g-X₅₅ -R-X₁₄ -h-X₃ (SEQ ID NO:17) (where invariantresidues are indicated by upper case letters and nearly invariantresidues are indicated by lower case letters). The nearly invariantresidues are usually found in most Ser/Thr kinase sites, but can bereplaced by other amino acids which, preferably, have similarcharacteristics. For example, a nearly invariant hydrophobic amino acidin the above amino acid consensus sequence would most likely be replacedby another hydrophobic amino acid. Ser/Thr kinase domains are describedin, for example, Levin D. E. et al. (1990) Proc. Natl. Acad. Sci. U.S.A87:8272-76, the contents of which are incorporated herein by reference.

As used herein, the term "ATP-binding region" includes an amino acidsequence of about 20-40, preferably 20-30, and more preferably 25-30amino acid residues in length, present in enzymes which activate theirsubstrates by phosphorylation, and involved in binding adenosinetriphosphate (ATP). ATP-binding regions preferably include the followingamino acid consensus sequence: G-X-G-X-X-G-X(15-23)-K(SEQ ID NO:18).ATP-binding regions are described in, for example, Samuel K. P. et al.(1987) FEBS Let. 218(1):81-86, the contents of which are incorporatedherein by reference. Amino acid residues 40 to 63 of CSAPK-1 comprise anATP-binding region.

Isolated proteins of the present invention, preferably CSAPK-1 proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:2 or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:1 or SEQ ID NO:3. As used herein,the term "sufficiently homologous" refers to a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains or motifs and/or acommon functional activity. For example, amino acid or nucleotidesequences which share common structural domains have at least 30%, 40%,or 50% homology, preferably 60% homology, more preferably 70%-80%, andeven more preferably 90-95% homology across the amino acid sequences ofthe domains and contain at least one and preferably two structuraldomains or motifs, are defined herein as sufficiently homologous.Furthermore, amino acid or nucleotide sequences which share at least30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95%homology and share a common functional activity are defined herein assufficiently homologous.

As used interchangeably herein a "CSAPK-1 activity", "biologicalactivity of CSAPK-1" or "functional activity of CSAPK-1", refers to anactivity exerted by a CSAPK-1 protein, polypeptide or nucleic acidmolecule on a CSAPK-1 responsive cell or a CSAPK-1 protein substrate, asdetermined in vivo, or in vitro, according to standard techniques. Thebiological activity of CSAPK-1 is described herein.

Accordingly, another embodiment of the invention features isolatedCSAPK-1 proteins and polypeptides having a CSAPK-1 activity. Preferredproteins are CSAPK-1 proteins having at least one Ser/Thr kinase siteand at least one ATP-binding region and, preferably, a CSAPK-1 activity.Additional preferred proteins have at least one Ser/Thr kinase site andat least one ATP-binding region and are, preferably, encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.

The nucleotide sequence of the isolated human CSAPK-1 cDNA and thepredicted amino acid sequence of the human CSAPK-1 polypeptide are shownin FIG. 1 and in SEQ ID NOs:1 and 2, respectively. A plasmid containingthe nucleotide sequence encoding human CSAPK-1 was deposited withAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, on Oct. 27, 1998 and assigned Accession Number203308. This deposit will be maintained under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

The CSAPK-1 gene, which is approximately 4137 nucleotides in length,encodes a protein having a molecular weight of approximately 34.7 kD andwhich is approximately 302 amino acid residues in length. The CSAPK-1gene is expressed predominantly in heart, skeletal muscle, or theplacenta.

B. The CSAPK-2 Nucleic Acid and Protein Molecules

In another embodiment, the isolated proteins of the present invention,preferably CSAPK-2 proteins, are identified based on the presence of atleast one dual specificity kinase catalytic domain and at least oneATP-binding region. In yet a further embodiment, the isolated proteinsof the present invention, preferably CSAPK-2 proteins, are identifiedbased on the presence of at least one dual specificity kinase catalyticdomain, at least one ATP-binding region, and at least one leucine zipperregion.

As used herein, the term "dual specificity kinase catalytic domain"includes an amino acid sequence of 200-400 amino acid residues inlength, preferably 200-300 amino acid residues in length, and morepreferably 250-300 amino acid residues in length, which includes akinase catalytic domain whose primary sequence is a hybrid between aserine/threonine kinase catalytic domain and a tyrosine kinase catalyticdomain. Kinases containing the dual specificity kinase catalytic domainare capable of phosphorylating both serine/threonine and tyrosineresidues. Preferably, a dual specificity kinase catalytic domainincludes the following amino acid consensus sequence X₉ -G-X-G-X₂-G-X-V-X₁₂ -K-X-.sub.(10-19) -E-X₃₄ -G-X₄₀ -H-R-D-X-K-X₂ -N-X₁₇ -K-X₂-D-F-G-X₁₉ -W-X-A-P-E-X₁₃ -W-X₇ -E-X₆ -P-X₃₆ -C-W-X₆ -R-P-X-F-X₁₄ (SEQID NO:19). Dual specificity kinase catalytic domains are described in,for example, Holzman L. B. et al. (1994) J. Biol. Chem. 269:30808-817,the contents of which are incorporated herein by reference. Amino acidresidues 31-277 of the CSAPK-2 protein comprise a dual specificitykinase catalytic domain.

As used herein, the term "leucine zipper region" includes a proteindomain which contains either leucine or isoleucine residues in everyseventh position over a stretch of at least 20-30 amino acid residues,more preferably at least 20-25 amino acid residues, and most preferablyat least 22 amino acid residues. Typically, the leucine zipper domain isrich in charged residues and forms a helical structure with ahydrophobic ridge of leucines or isoleucines down one face of the helix.The leucine zipper promotes dimerization through hydrophobicinteractions between the leucine residues. Leucine zipper domains aredescribed in, for example, Landschultz et al. (1988) Science 240:1759-1764, the contents of which are incorporated herein by reference.Amino acid residues 294 to 322 of the CSAPK-2 protein comprise a leucinezipper region. Optionally, the leucine zipper region is found adjacentto a basic region. As used herein, the term "basic region" includes anamino acid sequence of 5-30, preferably 5-20, more preferably 5-15 aminoacid residues in length, which is composed of a series of basic andhydrophobic amino acid residues, at either the N-terminus or theC-terminus of the leucine zipper region. For example, a 15 amino acidbasic domain can contain at least 6, 8, or 10 basic residues and/or atleast 1, 2, or 4 hydrophobic residues. Amino acid residues 407-421 ofthe CSAPK-2 protein comprise a basic region.

Isolated proteins of the present invention, preferably CSAPK-2 proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:5 or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:4 or SEQ ID NO:6.

As used interchangeably herein a "CSAPK-2 activity", "biologicalactivity of CSAPK-2" or "fuctional activity of CSAPK-2", refers to anactivity exerted by a CSAPK-2 protein, polypeptide or nucleic acidmolecule on a CSAPK-2 responsive cell or a CSAPK-2 protein substrate, asdetermined in vivo, or in vitro, according to standard techniques. Thebiological activity of CSAPK-2 is described herein.

Accordingly, another embodiment of the invention features isolatedCSAPK-2 proteins and polypeptides having a CSAPK-2 activity. Preferredproteins are CSAPK-2 proteins having at least one dual specificitykinase catalytic domain and at least one ATP-binding region. Yet furtherpreferred CSAPK-2 proteins have at least one dual specificity kinasecatalytic domain, at least one ATP-binding region, and at least oneleucine zipper region. Preferred proteins have at least one dualspecificity kinase catalytic domain, at least one ATP-binding regionand, preferably, a CSAPK-2 activity. Additional preferred proteins haveat least one dual specificity kinase catalytic domain, at least oneATP-binding region and are, preferably, encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:4 or SEQ ID NO:6.

The nucleotide sequence of the isolated human CSAPK-2 cDNA and thepredicted amino acid sequence of the human CSAPK-2 polypeptide are shownin FIG. 2 and in SEQ ID NOs:4 and 5, respectively. A plasmid containingthe nucleotide sequence encoding human CSAPK-2 was deposited withAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, on Oct. 27, 1998 and assigned Accession Number203306. This deposit will be maintained under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

The CSAPK-2 gene, which is approximately 2120 nucleotides in length,encodes a protein having a molecular weight of approximately 52.3 kD andwhich is approximately 455 amino acid residues in length. The CSAPK-2gene is expressed predominantly in muscle, e.g., skeletal or cardiacmuscle.

C. The CSAPK-3 Nucleic Acid and Protein Molecules

In another embodiment, the isolated proteins of the present invention,preferably CSAPK-3 proteins, are identified based on the presence of atleast one Ser/Thr kinase site and at least one ATP-binding region. TheSer/Thr kinase site and the ATP-binding region are described herein.Amino acid residues 5-164 of the CSAPK-3 protein comprise a Ser/Thrkinase domain.

Isolated proteins of the present invention, preferably CSAPK-3 proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:8 or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:7 or SEQ ID NO:9.

As used interchangeably herein a "CSAPK-3 activity", "biologicalactivity of CSAPK-3" or "functional activity of CSAPK-3", refers to anactivity exerted by a CSAPK-3 protein, polypeptide or nucleic acidmolecule on a CSAPK-3 responsive cell or a CSAPK-3 protein substrate asdetermined in vivo, or in vitro, according to standard techniques. Thebiological activity of CSAPK-3 is described herein.

Accordingly, another embodiment of the invention features isolatedCSAPK-3 proteins and polypeptides having a CSAPK-3 activity. Preferredproteins are CSAPK-3 proteins having at least one Ser/Thr kinase siteand at least one ATP-binding region. Additional preferred proteins haveat least one Ser/Thr kinase site, at least one ATP-binding region, and,preferably a CSAPK-3 activity. Additional preferred proteins have atleast one Ser/Thr kinase site and at least one ATP-binding region andare, preferably, encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:7or SEQ ID NO:9.

The nucleotide sequence of the isolated human CSAPK-3 cDNA and thepredicted amino acid sequence of the human CSAPK-3 polypeptide are shownin FIG. 3 and in SEQ ID NOs:7 and 8, respectively. A plasmid containingthe nucleotide sequence encoding human CSAPK-3 was deposited withAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, on Oct. 27, 1998 and assigned Accession Number203309. This deposit will be maintained under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

The CSAPK-3 gene, which is approximately 2454 nucleotides in length,encodes a protein having a molecular weight of approximately 66.8 kD andwhich is approximately 581 amino acid residues in length. The CSAPK-3gene is expressed predominantly in heart, skeletal muscle, brain,placenta, lung, liver, kidney, and pancreas.

D. The CSAPK-4 Nucleic Acid and Protein Molecules

In another embodiment, the isolated proteins of the present invention,preferably CSAPK-4 proteins, are identified based on the presence of atleast one Ser/Thr kinase site and at least one ATP-binding region. TheSer/Thr kinase site and the ATP-binding region are described herein.Amino acid residues 11 to 75 of the CSAPK-4 protein comprise a Ser/Thrkinase domain.

Isolated proteins of the present invention, preferably CSAPK-4 proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:11 or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:10 or SEQ ID NO:12.

As used interchangeably herein a "CSAPK-4 activity", "biologicalactivity of CSAPK-4" or "functional activity of CSAPK-4", refers to anactivity exerted by a CSAPK-4 protein, polypeptide or nucleic acidmolecule on a CSAPK-4 responsive cell or a CSAPK-4 protein substrate asdetermined in vivo, or in vitro, according to standard techniques. Thebiological activity of CSAPK-4 is described herein.

Accordingly, another embodiment of the invention features isolatedCSAPK-4 proteins and polypeptides having a CSAPK-4 activity. Preferredproteins are CSAPK-4 proteins having at least one Ser/Thr kinase siteand at least one ATP-binding region. Additional preferred proteins haveat least one Ser/Thr kinase site, at least one ATP-binding region, and,preferably a CSAPK-4 activity. Additional preferred proteins have atleast one Ser/Thr kinase site and at least one ATP-binding region andare, preferably, encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:10or SEQ ID NO:12.

The nucleotide sequence of the isolated human CSAPK-4 cDNA and thepredicted amino acid sequence of the human CSAPK-4 polypeptide are shownin FIG. 4 and in SEQ ID NOs:10 and 11, respectively. A plasmidcontaining the nucleotide sequence encoding human CSAPK-4 was depositedwith American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Oct. 27, 1998 and assignedAccession Number 203307. This deposit will be maintained under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure. This deposit wasmade merely as a convenience for those of skill in the art and is not anadmission that a deposit is required under 35 U.S.C. §112.

The human CSAPK-4 gene, which is approximately 1864 nucleotides inlength, encodes a protein having a molecular weight of approximately18.4 kD and which is approximately 160 amino acid residues in length.The CSAPK-4 gene is expressed predominantly in skeletal muscle.

E. The CSAPK-5 Nucleic Acid and Protein Molecules

In another embodiment, the isolated proteins of the present invention,preferably CSAPK-5 proteins, are identified based on the presence of atleast one Ser/Thr kinase site and at least one ATP-binding region. Inyet another embodiment, the isolated proteins of the present invention,preferably CSAPK-5 proteins, are identified based on the presence of atleast one Ser/Thr kinase site, and at least one ATP-binding region.

Isolated proteins of the present invention, preferably CSAPK-5 proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:14 or are encoded by a nucleotide sequencesufficiently homologous to SEQ ID NO:13 or SEQ ID NO:15.

As used interchangeably herein a "CSAPK-5 activity", "biologicalactivity of CSAPK-5" or "fuctional activity of CSAPK-5", refers to anactivity exerted by a CSAPK-5 protein, polypeptide or nucleic acidmolecule on a CSAPK-5 responsive cell or a CSAPK-5 protein substrate asdetermined in vivo, or in vitro, according to standard techniques. Thebiological activity of CSAPK-5 is described herein.

Accordingly, another embodiment of the invention features isolatedCSAPK-5 proteins and polypeptides having a CSAPK-5 activity. Preferredproteins are CSAPK-5 proteins having at least one Ser/Thr kinase siteand at least one ATP-binding region. Additional preferred proteins haveat least one Ser/Thr kinase site, at least one ATP-binding region, and,preferably a CSAPK-5 activity. Additional preferred proteins have atleast one Ser/Thr kinase site and at least one ATP-binding region andare, preferably, encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:13or SEQ ID NO:15.

The nucleotide sequence of the isolated human CSAPK-5 cDNA and thepredicted amino acid sequence of the human CSAPK-5 polypeptide are shownin FIG. 5 and in SEQ ID NOs:13 and 14, respectively. A plasmidcontaining the nucleotide sequence encoding human CSAPK-1 was depositedwith American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Oct. 27, 1998 and assignedAccession Number 203305. This deposit will be maintained under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure. This deposit wasmade merely as a convenience for those of skill in the art and is not anadmission that a deposit is required under 35 U.S.C. §112.

The human CSAPK-5 gene, which is approximately 1333 nucleotides inlength, encodes a protein having a molecular weight of approximately 51kD and which is approximately 444 amino acid residues in length.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode CSAPK proteins or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify CSAPK-encoding nucleic acids (e.g., CSAPK mRNA) andfragments for use as PCR primers for the amplification or mutation ofCSAPK nucleic acid molecules. As used herein, the term "nucleic acidmolecule" is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An "isolated" nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. For example, with regards to genomic DNA, the term"isolated" includes nucleic acid molecules which are separated from thechromosome with which the genomic DNA is naturally associated.Preferably, an "isolated" nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5' and3' ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated CSAPK nucleic acid molecule can contain less than about 5kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequenceswhich naturally flank the nucleic acid molecule in genomic DNA of thecell from which the nucleic acid is derived. Moreover, an "isolated"nucleic acid molecule, such as a cDNA molecule, can be substantiallyfree of other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:I0, SEQ IDNO:12, SEQ ID NO:13, or SEQ ID NO:15, or a portion thereof, can beisolated using standard molecular biology techniques and the sequenceinformation provided herein. For example, using all or portion of thenucleic acid sequence of SEQ ID NO:1, or the nucleotide sequence of SEQID NO:3, as a hybridization probe, CSAPK nucleic acid molecules can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:15 can beisolated by the polymerase chain reaction (PCR) using syntheticoligonucleotide primers designed based upon the sequence of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:15, respectively.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to CSAPK nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1. Thesequence of SEQ ID NO:1 corresponds to the partial human CSAPK-1 cDNA.This cDNA comprises sequences encoding the human CSAPK-1 protein (i.e.,"the coding region", from nucleotides 297-1202), as well as 5'untranslated sequences (nucleotides 1-296) and 3' untranslated sequences(nucleotides 1203-4137). Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:1 (e.g., nucleotides297-1202, corresponding to SEQ ID NO:3).

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:4. Thesequence of SEQ ID NO:4 corresponds to the partial human CSAPK-2 cDNA.This cDNA comprises sequences encoding the partial human CSAPK-2 protein(i.e., "the coding region", from nucleotides 47-1411), as well as 5'untranslated sequences (nucleotides 1-46) and 3' untranslated sequences(nucleotides 1412-2120). Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:4 (e.g., nucleotides47-1411, corresponding to SEQ ID NO:6).

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:7. Thesequence of SEQ ID NO:7 corresponds to the partial human CSAPK-3 cDNA.This cDNA comprises sequences encoding the partial human CSAPK-3 protein(i.e., "the coding region", from nucleotides 51-1793), as well as 5'untranslated sequences (nucleotides 1-50) and 3' untranslated sequences(nucleotides 1794-2454). Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:7 (e.g., nucleotides51-1793, corresponding to SEQ ID NO:9).

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:10. Thesequence of SEQ ID NO:10 corresponds to the partial human CSAPK-4 cDNA.This cDNA comprises sequences encoding the partial human CSAPK-4 protein(i.e., "the coding region", from nucleotides 275-754), as well as 5'untranslated sequences (nucleotides 1-274) and 3' untranslated sequences(nucleotides 755-1864). Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:10 (e.g., nucleotides275-754, corresponding to SEQ ID NO:12).

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:13. Thesequence of SEQ ID NO:13 corresponds to the partial human CSAPK-5 cDNA.This cDNA comprises sequences encoding the partial human CSAPK-5 protein(i.e., "the coding region", from nucleotides 2-1333).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:13, or SEQ ID NO:15, or a portion of any of these nucleotidesequences. A nucleic acid molecule which is complementary to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, or SEQ ID NO:15, is one which is sufficiently complementary tothe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:13, or SEQ ID NO:15, respectively, such that it can hybridize tothe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:13, or SEQ ID NO:15, respectively, thereby forming a stableduplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 54%, 55%, 60%, 62%, 65%, 70%, 75%, 78%, 80%, 85%, 86%,90%, 95%, 97%, 98% or more homologous to the nucleotide sequence (e.g.,to the entire length of the nucleotide sequence) shown in SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:15, or a portion of anyof these nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, or SEQ ID NO:15, for example a fragment which can be usedas a probe or primer or a fragment encoding a biologically activeportion of a CSAPK protein. The nucleotide sequence determined from thecloning of the CSAPK gene allows for the generation of probes andprimers designed for use in identifying and/or cloning other CSAPKfamily members, as well as CSAPK homologues from other species. Theprobe/primer typically comprises substantially purified oligonucleotide.The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:15, of ananti-sense sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, or SEQ ID NO:15, or of a naturally occurring allelic variant ormutant of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, or SEQ IDNO:15. In an exemplary embodiment, a nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at least 350,400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides in length andhybridizes under stringent hybridization conditions to a nucleic acidmolecule of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ IDNO:15.

Probes based on the CSAPK nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissues which misexpress a CSAPK protein, such as by measuring a levelof a CSAPK-encoding nucleic acid in a sample of cells from a subjecte.g., detecting CSAPK mRNA levels or determining whether a genomic CSAPKgene has been mutated or deleted.

A nucleic acid fragment encoding a "biologically active portion of aCSAPK protein" can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ IDNO:15, which encodes a polypeptide having a CSAPK biological activity(the biological activities of the CSAPK proteins are described herein),expressing the encoded portion of the CSAPK protein (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of the CSAPK protein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, or SEQ ID NO:15, due to the degeneracy of the genetic codeand, thus, encode the same CSAPK proteins as those encoded by thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, or SEQ ID NO:15. In another embodiment, an isolated nucleic acidmolecule of the invention has a nucleotide sequence encoding a proteinhaving an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:11, or SEQ ID NO:14.

In addition to the CSAPK nucleotide sequences shown in SEQ ID NO:1, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:15, it will beappreciated by those skilled in the art that DNA sequence polymorphismsthat lead to changes in the amino acid sequences of the CSAPK proteinsmay exist within a population (e.g., the human population). Such geneticpolymorphism in the CSAPK genes may exist among individuals within apopulation due to natural allelic variation. As used herein, the terms"gene" and "recombinant gene" refer to nucleic acid molecules whichinclude an open reading frame encoding an CSAPK protein, preferably amammalian CSAPK protein, and can further include non-coding regulatorysequences, and introns. Such natural allelic variations include bothfunctional and non-functional CSAPK proteins and can typically result in1-5% variance in the nucleotide sequence of a CSAPK gene. Any and allsuch nucleotide variations and resulting amino acid polymorphisms inCSAPK genes that are the result of natural allelic variation and that donot alter the functional activity of a CSAPK protein are intended to bewithin the scope of the invention.

Moreover, nucleic acid molecules encoding other CSAPK family membersand, thus, which have a nucleotide sequence which differs from the CSAPKsequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ IDNO:15 are intended to be within the scope of the invention. For example,another CSAPK cDNA can be identified based on the nucleotide sequence ofhuman CSAPK. Moreover, nucleic acid molecules encoding CSAPK proteinsfrom different species, and thus which have a nucleotide sequence whichdiffers from the CSAPK sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, or SEQ ID NO:15 are intended to be within the scope of theinvention. For example, a mouse CSAPK cDNA can be identified based onthe nucleotide sequence of a human CSAPK.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the CSAPK cDNAs of the invention can be isolated based ontheir homology to the CSAPK nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, or SEQ ID NO:15. In other embodiment, the nucleic acid isat least 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or600 nucleotides in length. As used herein, the term "hybridizes understringent conditions" is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 30%,40%, 50%, or 60% homologous to each other typically remain hybridized toeach other. Preferably, the conditions are such that sequences at leastabout 70%, more preferably at least about 80%, even more preferably atleast about 85% or 90% homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, New York (1989), 6.3.1-6.3.6. A preferred,non-limiting example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:15 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a"naturally-occurring" nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

In addition to naturally-occurring allelic variants of the CSAPKsequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, or SEQ ID NO:15, thereby leading to changes in the amino acidsequence of the encoded CSAPK proteins, without altering the functionalability of the CSAPK proteins. For example, nucleotide substitutionsleading to amino acid substitutions at "non-essential" amino acidresidues can be made in the sequence oSEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, or SEQ ID NO:15. A "non-essential" amino acid residue is aresidue that can be altered from the wild-type sequence of CSAPK (e.g.,the sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, orSEQ ID NO:14) without altering the biological activity, whereas an"essential" amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the CSAPK proteinsof the present invention, are predicted to be particularly unamenable toalteration. Furthermore, additional amino acid residues that areconserved between the CSAPK proteins of the present invention and otherCSAPK family members are not likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding CSAPK proteins that contain changes in amino acidresidues that are not essential for activity. Such CSAPK proteins differin amino acid sequence from SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQID NO:11, or SEQ ID NO:14, yet retain biological activity. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein comprises an amino acidsequence at least about 41%, 42%, 45%, 50%, 55%, 59%, 60%, 65%, 70%,75%, 80% 81%, 85%, 90%, 95%, 98% or more homologous to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQID NO:14 (e.g., the entire amino acid sequence of SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:14).

An isolated nucleic acid molecule encoding a CSAPK protein homologous tothe protein of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, orSEQ ID NO:14 can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofSEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:15,respectively, such that one or more amino acid substitutions, additionsor deletions are introduced into the encoded protein. Mutations can beintroduced into SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, orSEQ ID NO:15 by standard techniques, such as site-directed mutagenesisand PCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A "conservative amino acid substitution" is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a CSAPKprotein is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a CSAPK coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for CSAPK biological activity to identify mutants that retainactivity. Following mutagenesis of SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:10, or SEQ ID NO:15, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined.

In a preferred embodiment, a mutant CSAPK protein can be assayed for theability to: 1) regulate trasmission of signals from cellular receptors,e.g., cardiac cell growth factor receptors; 2) control entry of cells,e.g., cardiac cells, into mitosis; 3) modulate cellular differentiation;4) modulate cell death; or 5) regulate cytoskeleton function, e.g.,actin bundling.

In addition to the nucleic acid molecules encoding CSAPK proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An "antisense"nucleic acid comprises a nucleotide sequence which is complementary to a"sense" nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire CSAPK coding strand, or only to a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a "codingregion" of the coding strand of a nucleotide sequence encoding CSAPK.The term "coding region" refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the coding region of human CSAPK-1 corresponds to SEQ ID NO:3). Inanother embodiment, the antisense nucleic acid molecule is antisense toa "noncoding region" of the coding strand of a nucleotide sequenceencoding CSAPK. The term "noncoding region" refers to 5' and 3'sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5' and 3' untranslated regions).

Given the coding strand sequences encoding CSAPK disclosed herein (e.g.,SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, or SEQ ID NO:15),antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of CSAPK mRNA,but more preferably is an oligonucleotide which is antisense to only aportion of the coding or noncoding region of CSAPK mRNA. For example,the antisense oligonucleotide can be complementary to the regionsurrounding the translation start site of CSAPK mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in length. An antisense nucleic acid of theinvention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylarninomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5'-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a CSAPKprotein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hanmmerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveCSAPK mRNA transcripts to thereby inhibit translation of CSAPK mRNA. Aribozyme having specificity for a CSAPK-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a CSAPK cDNA disclosedherein (i.e., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ IDNO:15). For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in aCSAPK-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; andCech et al. U.S. Pat. No. 5,116,742. Alternatively, CSAPK mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W.(1993) Science 261:1411-1418.

Alternatively, CSAPK gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the CSAPK(e.g., the CSAPK promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the CSAPK gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

In yet another embodiment, the CSAPK nucleic acid molecules of thepresent invention can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & MedicinalChemistry 4 (1): 5-23). As used herein, the terms "peptide nucleicacids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, inwhich the deoxyribose phosphate backbone is replaced by a pseudopeptidebackbone and only the four natural nucleobases are retained. The neutralbackbone of PNAs has been shown to allow for specific hybridization toDNA and RNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe etal. Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of CSAPK nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of CSAPK nucleic acid molecules can also be used inthe analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as `artificial restriction enzymes` whenused in combination with other enzymes, (e.g., S1 nucleases (Hyrup B.(1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In another embodiment, PNAs of CSAPK can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of CSAPK nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup B. (1996) supra and Finn P. J. et al.(1996) Nucleic Acids Res. 24 (17):3357-63. For example, a DNA chain canbe synthesized on a solid support using standard phosphoramiditecoupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be usedas a between the PNA and the 5' end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5' PNA segment anda 3' DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

II. Isolated CSAPK Proteins and Anti-CSAPK Antibodies

One aspect of the invention pertains to isolated CSAPK proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-CSAPK antibodies. In oneembodiment, native CSAPK proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, CSAPK proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a CSAPK protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

An "isolated" or "purified" protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theCSAPK protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language"substantially free of cellular material" includes preparations of CSAPKprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language "substantially free of cellular material"includes preparations of CSAPK protein having less than about 30% (bydry weight) of non-CSAPK protein (also referred to herein as a"contaminating protein"), more preferably less than about 20% ofnon-CSAPK protein, still more preferably less than about 10% ofnon-CSAPK protein, and most preferably less than about 5% non-CSAPKprotein. When the CSAPK protein or biologically active portion thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

The language "substantially free of chemical precursors or otherchemicals" includes preparations of CSAPK protein in which the proteinis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. In one embodiment, thelanguage "substantially free of chemical precursors or other chemicals"includes preparations of CSAPK protein having less than about 30% (bydry weight) of chemical precursors or non-CSAPK chemicals, morepreferably less than about 20% chemical precursors or non-CSAPKchemicals, still more preferably less than about 10% chemical precursorsor non-CSAPK chemicals, and most preferably less than about 5% chemicalprecursors or non-CSAPK chemicals.

Biologically active portions of a CSAPK protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the CSAPK protein, e.g., the amino acidsequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11,or SEQ ID NO:14, which include less amino acids than the full lengthCSAPK proteins, and exhibit at least one activity of a CSAPK protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the CSAPK protein. A biologically activeportion of a CSAPK protein can be a polypeptide which is, for example,at least 10, 25, 50, 100 or more amino acids in length.

In a preferred embodiment, the CSAPK protein has an amino acid sequenceshown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQ IDNO:14. In other embodiments, the CSAPK protein is substantiallyhomologous to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, orSEQ ID NO:14, and retains the functional activity of the protein of SEQID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:14, yetdiffers in amino acid sequence due to natural allelic variation ormutagenesis, as described in detail in subsection I above. Accordingly,in another embodiment, the CSAPK protein is a protein which comprises anamino acid sequence at least about 41%, 42%, 45%, 50%, 55%, 59%, 60%,65%, 70%, 75%, 80% 81%, 85%, 90%, 95%, 98% or more homologous to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:11, or SEQ ID NO:14 (e.g., the entire amino acid sequence of SEQ IDNO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:14).

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the CSAPK amino acidsequence of SEQ ID NO:2, 5, 8, 11, or 14 having 177 amino acid residues,at least 80, preferably at least 100, more preferably at least 120, evenmore preferably at least 140, and even more preferably at least 150, 160or 170 amino acid residues are aligned). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid"identity" is equivalent to amino acid or nucleic acid "homology"). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the GAP program in the GCGsoftware package (available at http://www.gcg.com), using either aBlossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yetanother preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a "query sequence" to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to CSAPK nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to CSAPK proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The invention also provides CSAPK chimeric or fusion proteins. As usedherein, a CSAPK "chimeric protein" or "fusion protein" comprises a CSAPKpolypeptide operatively linked to a non-CSAPK polypeptide. An "CSAPKpolypeptide" refers to a polypeptide having an amino acid sequencecorresponding to CSAPK, whereas a "non-CSAPK polypeptide" refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the CSAPK protein, e.g., aprotein which is different from the CSAPK protein and which is derivedfrom the same or a different organism. Within a CSAPK fusion protein theCSAPK polypeptide can correspond to all or a portion of a CSAPK protein.In a preferred embodiment, a CSAPK fusion protein comprises at least onebiologically active portion of a CSAPK protein. In another preferredembodiment, a CSAPK fusion protein comprises at least two biologicallyactive portions of a CSAPK protein. Within the fusion protein, the term"operatively linked" is intended to indicate that the CSAPK polypeptideand the non-CSAPK polypeptide are fused in-frame to each other. Thenon-CSAPK polypeptide can be fused to the N-terminus or C-terminus ofthe CSAPK polypeptide.

For example, in one embodiment, the fusion protein is a GST-CSAPK fusionprotein in which the CSAPK sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant CSAPK.

In another embodiment, the fusion protein is a CSAPK protein containinga heterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of CSAPK canbe increased through use of a heterologous signal sequence.

The CSAPK fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. TheCSAPK fusion proteins can be used to affect the bioavailability of aCSAPK substrate. Use of CSAPK fusion proteins may be usefultherapeutically for the treatment of cellular growth related disorders,e.g., cardiovascular disorers. Moreover, the CSAPK-fusion proteins ofthe invention can be used as immunogens to produce anti-CSAPK antibodiesin a subject, to purify CSAPK ligands and in screening assays toidentify molecules which inhibit the interaction of CSAPK with a CSAPKsubstrate.

Preferably, a CSAPK chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). ACSAPK-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the CSAPK protein.

The present invention also pertains to variants of the CSAPK proteinswhich function as either CSAPK agonists (mimetics) or as CSAPKantagonists. Variants of the CSAPK proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of a CSAPKprotein. An agonist of the CSAPK proteins can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of a CSAPK protein. An antagonist of a CSAPK protein caninhibit one or more of the activities of the naturally occurring form ofthe CSAPK protein by, for example, competitively modulating acardiovascular system activity of a CSAPK protein. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. In one embodiment, treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the CSAPKprotein.

In one embodiment, variants of a CSAPK protein which function as eitherCSAPK agonists (mimetics) or as CSAPK antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a CSAPK protein for CSAPK protein agonist or antagonist activity. Inone embodiment, a variegated library of CSAPK variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of CSAPK variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential CSAPK sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of CSAPK sequences therein. There are avariety of methods which can be used to produce libraries of potentialCSAPK variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential CSAPK sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477.

In addition, libraries of fragments of a CSAPK protein coding sequencecan be used to generate a variegated population of CSAPK fragments forscreening and subsequent selection of variants of a CSAPK protein. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double stranded PCR fragment of a CSAPK coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double stranded DNA, renaturing the DNA toform double stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the CSAPK protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of CSAPK proteins. The mostwidely used techniques, which are amenable to high through-out analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recrusive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of fimctional mutants in thelibraries, can be used in combination with the screening assays toidentify CSAPK variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated CSAPK library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily synthesizes andsecretes CSAPK. The transfected cells are then cultured such that CSAPKand a particular mutant CSAPK are secreted and the effect of expressionof the mutant on CSAPK activity in cell supernatants can be detected,e.g., by any of a number of enzymatic assays. Plasmid DNA can then berecovered from the cells which score for inhibition, or alternatively,potentiation of CSAPK activity, and the individual clones furthercharacterized.

An isolated CSAPK protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind CSAPK using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length CSAPK protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of CSAPK for use as immunogens. Theantigenic peptide of CSAPK comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8,SEQ ID NO:11, or SEQ ID NO:14 and encompasses an epitope of CSAPK suchthat an antibody raised against the peptide forms a specific immunecomplex with CSAPK. Preferably, the antigenic peptide comprises at least10 amino acid residues, more preferably at least 15 amino acid residues,even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofCSAPK that are located on the surface of the protein, e.g., hydrophilicregions.

A CSAPK immunogen typically is used to prepare antibodies by immunizinga suitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed CSAPK protein or a chemicallysynthesized CSAPK polypeptide. The preparation can firther include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic CSAPK preparation induces a polyclonal anti-CSAPK antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-CSAPKantibodies. The term "antibody" as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as CSAPK.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab')₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind CSAPK. The term"monoclonal antibody" or "monoclonal antibody composition", as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of CSAPK. A monoclonal antibody composition thustypically displays a single binding affinity for a particular CSAPKprotein with which it immunoreacts.

Polyclonal anti-CSAPK antibodies can be prepared as described above byimmunizing a suitable subject with a CSAPK immunogen. The anti-CSAPKantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized CSAPK. If desired, the antibody moleculesdirected against CSAPK can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-CSAPK antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique(Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J Biol. Med., 54:387-402; M. L. Gefter et al. (1977) SomaticCell Genet. 3:231-36). Briefly, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a CSAPK immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds CSAPK.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-CSAPK monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol ("PEG"). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindCSAPK, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-CSAPK antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with CSAPK to thereby isolateimmunoglobulin library members that bind CSAPK. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol.Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram etal. (1992) Proc. Natl. Acad Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-CSAPK antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad Sci. USA 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-CSAPK antibody (e.g., monoclonal antibody) can be used toisolate CSAPK by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-CSAPK antibody can facilitate thepurification of natural CSAPK from cells and of recombinantly producedCSAPK expressed in host cells. Moreover, an anti-CSAPK antibody can beused to detect CSAPK protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the CSAPK protein. Anti-CSAPK antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, -galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a CSAPK protein(or a portion thereof). As used herein, the term "vector" refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a "plasmid", whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated.

Another type of vector is a viral vector, wherein additional DNAsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as "expression vectors". In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, "plasmid" and"vector" can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, "operably linked" is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term "regulatory sequence" isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., CSAPK proteins,mutant forms of CSAPK proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of CSAPK proteins in prokaryotic or eukaryotic cells. Forexample, CSAPK proteins can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in CSAPK activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific for CSAPK proteins, for example. In apreferred embodiment, a CSAPK fusion protein expressed in a retroviralexpression vector of the present invention can be utilized to infectbone marrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time has passed (e.g., six (6) weeks).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS1174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the CSAPK expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1(Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(InVitrogen Corp, San Diego, Calif.).

Alternatively, CSAPK proteins can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to CSAPK mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews--Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms "host cell" and "recombinant host cell" are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aCSAPK protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms "transformation" and "transfection" are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a CSAPK protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a CSAPK protein.Accordingly, the invention further provides methods for producing aCSAPK protein using the host cells of the invention. In one embodiment,the method comprises culturing the host cell of invention (into which arecombinant expression vector encoding a CSAPK protein has beenintroduced) in a suitable medium such that a CSAPK protein is produced.In another embodiment, the method further comprises isolating a CSAPKprotein from the medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichCSAPK-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous CSAPKsequences have been introduced into their genome or homologousrecombinant animals in which endogenous CSAPK sequences have beenaltered. Such animals are useful for studying the function and/oractivity of a CSAPK and for identifying and/or evaluating modulators ofCSAPK activity. As used herein, a "transgenic animal" is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a "homologous recombinant animal" isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous CSAPK gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing aCSAPK-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The CSAPKcDNA sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, orSEQ ID NO:13 can be introduced as a transgene into the genome of anon-human animal. Alternatively, a nonhuman homologue of a human CSAPKgene, such as a mouse or rat CSAPK gene, can be used as a transgene.Alternatively, a CSAPK gene homologue, such as another CSAPK familymember, can be isolated based on hybridization to the CSAPK cDNAsequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, or SEQ IDNO:15 (described further in subsection I above) and used as a transgene.Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to aCSAPK transgene to direct expression of a CSAPK protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a CSAPK transgene in its genome and/or expression of CSAPKmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding a CSAPKprotein can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a CSAPK gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the CSAPK gene. The CSAPK gene can be a human gene(e.g., the SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQID NO:13), but more preferably, is a non-human homologue of a humanCSAPK gene (e.g., a cDNA isolated by stringent hybridization with thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ IDNO:10, or SEQ ID NO:13). For example, a mouse CSAPK gene can be used toconstruct a homologous recombination vector suitable for altering anendogenous CSAPK gene in the mouse genome. In a preferred embodiment,the vector is designed such that, upon homologous recombination, theendogenous CSAPK gene is functionally disrupted (i.e., no longer encodesa functional protein; also referred to as a "knock out" vector).Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous CSAPK gene is mutated or otherwise alteredbut still encodes a functional protein (e.g., the upstream regulatoryregion can be altered to thereby alter the expression of the endogenousCSAPK protein). In the homologous recombination vector, the alteredportion of the CSAPK gene is flanked at its 5' and 3' ends by additionalnucleic acid sequence of the CSAPK gene to allow for homologousrecombination to occur between the exogenous CSAPK gene carried by thevector and an endogenous CSAPK gene in an embryonic stem cell. Theadditional flanking CSAPK nucleic acid sequence is of sufficient lengthfor successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5' and 3'ends) are included in the vector (see e.g., Thomas, K. R. and Capecchi,M. R. (1987) Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced CSAPK genehas homologously recombined with the endogenous CSAPK gene are selected(see, e.g., Li, E. et al. (1992) Cell 69:915). The selected cells arethen injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of "double"transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The recontructed oocyte is then cultured such that it develops to morulaor blastocyte and then transferred to pseudopregnant female fosteranimal. The offspring borne of this female foster animal will be a cloneof the animal from which the cell, e.g., the somatic cell, is isolated.

IV. Pharmaceutical Compositions

The CSAPK nucleic acid molecules, CSAPK proteins, and anti-CSAPKantibodies (also referred to herein as "active compounds") of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language "pharmaceutically acceptablecarrier" is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a CSAPK protein or anti-CSAPK antibody) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Theisolated nucleic acid molecules of the invention can be used, forexample, to express CSAPK protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect CSAPKmRNA (e.g., in a biological sample) or a genetic alteration in a CSAPKgene, and to modulate CSAPK activity, as described further below. TheCSAPK proteins can be used to treat disorders characterized byinsufficient or excessive production of a CSAPK substrate or productionof CSAPK inhibitors. In addition, the CSAPK proteins can be used toscreen for naturally occurring CSAPK substrates, to screen for drugs orcompounds which modulate CSAPK activity, as well as to treat disorderscharacterized by insufficient or excessive production of CSAPK proteinor production of CSAPK protein forms which have decreased or aberrantactivity compared to CSAPK wild type protein. Moreover, the anti-CSAPKantibodies of the invention can be used to detect and isolate CSAPKproteins, regulate the bioavailability of CSAPK proteins, and modulateCSAPK activity.

A. Screening Assays:

The invention provides a method (also referred to herein as a "screeningassay") for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to CSAPK proteins, have a stimulatory or inhibitory effecton, for example, CSAPK expression or CSAPK activity, or have astimulatory or inhibitory effect on, for example, the expression oractivity of a CSAPK substrate.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a CSAPK protein or polypeptideor biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of a CSAPK protein or polypeptideor biologically active portion thereof, e.g., modulate the ability ofCSAPK to interact with its cognate ligand. The test compounds of thepresent invention can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the`one-bead one-compound` library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. AcadSci. USA. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a CSAPK target molecule (e.g., a CSAPKphosphorylation substrate) with a test compound and determining theability of the test compound to modulate (e.g. stimulate or inhibit) theactivity of the CSAPK target molecule. Determining the ability of thetest compound to modulate the activity of a CSAPK target molecule can beaccomplished, for example, by determining the ability of the CSAPKprotein to bind to or interact with the CSAPK target molecule, or bydetermining the ability of the CSAPK protein to phosphorylate the CSAPKtarget molecule.

The ability of the CSAPK protein to phosphorylate a CSAPK targetmolecule can be determined by, for example, an in vitro kinase assay.Briefly, a CSAPK target molecule, e.g., an immunoprecipitated CSAPKtarget molecule from a cell line expressing such a molecule, can beincubated with the CSAPK protein and radioactive ATP, e.g., [γ-³² P]ATP, in a buffer containing MgCl₂ and MnCl₂, e.g., 10 mM MgCl₂ and 5 mMMnCl₂. Following the incubation, the immunoprecipitated CSAPK targetmolecule can be separated by SDS-polyacrylamide gel electrophoresisunder reducing conditions, transferred to a membrane, e.g., a PVDFmembrane, and autoradiographed. The appearance of detectable bands onthe autoradiograph indicates that the CSAPK substrate has beenphosphorylated. Phosphoaminoacid analysis of the phosphorylatedsubstrate can also be performed in order to determine which residues onthe CSAPK substrate are phosphorylated. Briefly, the radiophosphorylatedprotein band can be excised from the SDS gel and subjected to partialacid hydrolysis. The products can then be separated by one-dimensionalelectrophoresis and analyzed on, for example, a phosphoimager andcompared to ninhydrin-stained phosphoaminoacid standards.

Determining the ability of the CSAPK protein to bind to or interact witha CSAPK target molecule can be accomplished by determining directbinding. Determining the ability of the CSAPK protein to bind to orinteract with a CSAPK target molecule can be accomplished, for example,by coupling the CSAPK protein with a radioisotope or enzymatic labelsuch that binding of the CSAPK protein to a CSAPK target molecule can bedetermined by detecting the labeled CSAPK protein in a complex. Forexample, CSAPK molecules, e.g., CSAPK proteins, can be labeled with ¹²⁵I, ³⁵ S, ¹⁴ C, or ³ H, either directly or indirectly, and theradioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, CSAPK molecules can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound to modulate the interaction between CSAPK and its targetmolecule, without the labeling of any of the interactants. For example,a microphysiometer can be used to detect the interaction of CSAPK withits target molecule without the labeling of either CSAPK or the targetmolecule. McConnell, H. M. et al. (1992) Science 257:1906-1912. As usedherein, a "microphysiometer" (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between compound and receptor.

In a preferred embodiment, determining the ability of the CSAPK proteinto bind to or interact with a CSAPK target molecule can be accomplishedby determining the activity of the target molecule. For example, theactivity of the target molecule can be determined by detecting inductionof a cellular second messenger of the target (e.g., intracellular Ca²⁺,diacylglycerol, IP₃, etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of areporter gene (comprising a target-responsive regulatory elementoperatively linked to a nucleic acid encoding a detectable marker, e.g.,chloramphenicol acetyl transferase), or detecting a target-regulatedcellular response.

In yet another embodiment, an assay of the present invention is acell-free assay in which a CSAPK protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to the CSAPK protein or biologically active portionthereof is determined. Binding of the test compound to the CSAPK proteincan be determined either directly or indirectly as described above. In apreferred embodiment, the assay includes contacting the CSAPK protein orbiologically active portion thereof with a known compound which bindsCSAPK to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a CSAPK protein, wherein determining the ability of the testcompound to interact with a CSAPK protein comprises determining theability of the test compound to preferentially bind to CSAPK orbiologically active portion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a CSAPKprotein or biologically active portion thereof is contacted with a testcompound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the CSAPK protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a CSAPK protein can beaccomplished, for example, by determining the ability of the CSAPKprotein to bind to a CSAPK target molecule by one of the methodsdescribed above for determining direct binding. Determining the abilityof the CSAPK protein to bind to a CSAPK target molecule can also beaccomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705. As used herein, "BIA" is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BlAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a CSAPK protein can be accomplishedby determining the ability of the CSAPK protein to further modulate theactivity of a CSAPK target molecule (e.g., a CSAPK mediated signaltransduction pathway component). For example, the activity of theeffector molecule on an appropriate target can be determined, or thebinding of the effector to an appropriate target can be determined aspreviously described.

In yet another embodiment, the cell-free assay involves contacting aCSAPK protein or biologically active portion thereof with a knowncompound which binds the CSAPK protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the CSAPK protein, whereindetermining the ability of the test compound to interact with the CSAPKprotein comprises determining the ability of the CSAPK protein topreferentially bind to or modulate the activity of a CSAPK targetmolecule.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of proteins (e.g., CSAPKproteins or biologically active portions thereof, or receptors to whichCSAPK binds). In the case of cell-free assays in which a membrane-boundform a protein is used (e.g., a cell surface CSAPK receptor) it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of the protein is maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either CSAPK or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to a CSAPK protein, or interaction ofa CSAPK protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/CSAPK fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or CSAPK protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of CSAPKbinding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a CSAPKprotein or a CSAPK target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated CSAPK protein ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with CSAPK protein or target molecules but which donot interfere with binding of the CSAPK protein to its target moleculecan be derivatized to the wells of the plate, and unbound target orCSAPK protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the CSAPK protein or target molecule, as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the CSAPK protein or target molecule.

In another embodiment, modulators of CSAPK expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of CSAPK mRNA or protein in the cell is determined. The levelof expression of CSAPK mRNA or protein in the presence of the candidatecompound is compared to the level of expression of CSAPK mRNA or proteinin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of CSAPK expression based on thiscomparison. For example, when expression of CSAPK mRNA or protein isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of CSAPK mRNA or protein expression.Alternatively, when expression of CSAPK mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of CSAPK mRNA or protein expression. The level of CSAPK mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting CSAPK mRNA or protein.

In yet another aspect of the invention, the CSAPK proteins can be usedas "bait proteins" in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with CSAPK ("CSAPK-binding proteins" or "CSAPK-bp")and are involved in CSAPK activity. Such CSAPK-binding proteins are alsolikely to be involved in the propagation of signals by the CSAPKproteins or CSAPK targets as, for exarnple, downstream elements of aCSAPK-mediated signaling pathway. Alternatively, such CSAPK-bindingproteins are likely to be CSAPK inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a CSAPK protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein ("prey"or "sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. If the "bait" and the "prey" proteinsare able to interact, in vivo, forming a CSAPK-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the CSAPKprotein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a CSAPK modulating agent, an antisense CSAPKnucleic acid molecule, a CSAPK-specific antibody, or a CSAPK-bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the CSAPK nucleotide sequences, describedherein, can be used to map the location of the CSAPK genes on achromosome. The mapping of the CSAPK sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, CSAPK genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the CSAPK nucleotidesequences. Computer analysis of the CSAPK sequences can be used topredict primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhwnan chromosomes. Only those hybrids containing the human genecorresponding to the CSAPK sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the CSAPKnucleotide sequences to design oligonucleotide primers, sublocalizationcan be achieved with panels of fragments from specific chromosomes.Other mapping strategies which can similarly be used to map a 9o, 1p, or1v sequence to its chromosome include in situ hybridization (describedin Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the CSAPK gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The CSAPK sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of "Dog Tags" which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the CSAPK nucleotide sequences described herein can beused to prepare two PCR primers from the 5' and 3' ends of thesequences. These primers can then be used to amplify an individual's DNAand subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The CSAPK nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1, SEQID NO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:13, can comfortablyprovide positive individual identification with a panel of perhaps 10 to1,000 primers which each yield a noncoding amplified sequence of 100bases. If predicted coding sequences, such as those in SEQ ID NO:3, SEQID NO:6, SEQ ID NO:9, SEQ ID NO:12, or SEQ ID NO:15 are used, a moreappropriate number of primers for positive individual identificationwould be 500-2,000.

If a panel of reagents from CSAPK nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of Partial CSAPK Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another"identification marker" (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7,SEQ ID NO:10, or SEQ ID NO:13 are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the CSAPK nucleotidesequences or portions thereof, e.g., fragments derived from thenoncoding regions of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ IDNO:10, or SEQ ID NO:13, having a length of at least 20 bases, preferablyat least 30 bases.

The CSAPK nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue.

This can be very useful in cases where a forensic pathologist ispresented with a tissue of unknown origin. Panels of such CSAPK probescan be used to identify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., CSAPK primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining CSAPK proteinand/or nucleic acid expression as well as CSAPK activity, in the contextof a biological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant CSAPKexpression or activity. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping a disorder associated with CSAPK protein, nucleic acidexpression or activity. For example, mutations in a CSAPK gene can beassayed in a biological sample. Such assays can be used for prognosticor predictive purpose to thereby phophylactically treat an individualprior to the onset of a disorder characterized by or associated withCSAPK protein, nucleic acid expression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of CSAPKin clinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of CSAPKprotein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting CSAPK protein ornucleic acid (e.g., mRNA, genomic DNA) that encodes CSAPK protein suchthat the presence of CSAPK protein or nucleic acid is detected in thebiological sample. A preferred agent for detecting CSAPK mRNA or genomicDNA is a labeled nucleic acid probe capable of hybridizing to CSAPK mRNAor genomic DNA. The nucleic acid probe can be, for example, a humanCSAPK nucleic acid, such as the nucleic acid of SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:13, or a portion thereof,such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to CSAPK mRNA or genomic DNA. Other suitable probesfor use in the diagnostic assays of the invention are described herein.

A preferred agent for detecting CSAPK protein is an antibody capable ofbinding to CSAPK protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can beused. The term "labeled", with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The term "biological sample" is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect CSAPK mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of CSAPK mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of CSAPK protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of CSAPK genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of CSAPK protein include introducing into a subject a labeledanti-CSAPK antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting CSAPK protein, mRNA, orgenomic DNA, such that the presence of CSAPK protein, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofCSAPK protein, mRNA or genomic DNA in the control sample with thepresence of CSAPK protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of CSAPKin a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting CSAPK protein or mRNA in abiological sample; means for determining the amount of CSAPK in thesample; and means for comparing the amount of CSAPK in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectCSAPK protein or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant CSAPK expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with CSAPK protein, nucleicacid expression or activity. Thus, the present invention provides amethod for identifying a disease or disorder associated with aberrantCSAPK expression or activity in which a test sample is obtained from asubject and CSAPK protein or nucleic acid (e.g., mRNA, genomic DNA) isdetected, wherein the presence of CSAPK protein or nucleic acid isdiagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant CSAPK expression or activity. As usedherein, a "test sample" refers to a biological sample obtained from asubject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant CSAPK expression or activity. Thus, the presentinvention provides methods for determining whether a subject can beeffectively treated with an agent for a disorder associated withaberrant CSAPK expression or activity in which a test sample is obtainedand CSAPK protein or nucleic acid expression or activity is detected(e.g., wherein the abundance of CSAPK protein or nucleic acid expressionor activity is diagnostic for a subject that can be administered theagent to treat a disorder associated with aberrant CSAPK expression oractivity).

The methods of the invention can also be used to detect geneticalterations in a CSAPK gene, thereby determining if a subject with thealtered gene is at risk for a disorder associated with the CSAPK gene.In preferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a CSAPK-protein, or the mis-expression of the CSAPKgene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a CSAPK gene; 2) an addition of one or morenucleotides to a CSAPK gene; 3) a substitution of one or morenucleotides of a CSAPK gene, 4) a chromosomal rearrangement of a CSAPKgene; 5) an alteration in the level of a messenger RNA transcript of aCSAPK gene, 6) aberrant modification of a CSAPK gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a CSAPK gene, 8)a non-wild type level of a CSAPK-protein, 9) allelic loss of a CSAPKgene, and 10) inappropriate post-translational modification of aCSAPK-protein. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting alterationsin a CSAPK gene. A preferred biological sample is a tissue or serumsample isolated by conventional means from a subject, e.g., a cardiactissue sample.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad Sci. USA 91:360-364), the latter of which can be particularlyuseful for detecting point mutations in the CSAPK-gene (see Abravaya etal. (1995) Nucleic Acids Res .23:675-682). This method can include thesteps of collecting a sample of cells from a patient, isolating nucleicacid (e.g., genomic, mRNA or both) from the cells of the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a CSAPK gene under conditions such thathybridization and amplification of the CSAPK-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a CSAPK gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in CSAPK can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in CSAPK can be identified in two dimensional arrayscontaining light-generated DNA probes as described in Cronin, M. T. etal. supra. Briefly, a first hybridization array of probes can be used toscan through long stretches of DNA in a sample and control to identifybase changes between the sequences by making linear arrays of sequentialovelapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the CSAPK gene anddetect mutations by comparing the sequence of the sample CSAPK with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad Sci. USA 74:560) or Sanger ((1977) inProc. Natl Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the CSAPK gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of "mismatch cleavage" startsby providing heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type CSAPK sequence with potentially mutant RNA orDNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control DNA or RNA can belabeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called "DNA mismatch repair" enzymes) in defined systems fordetecting and mapping point mutations in CSAPK cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a CSAPK sequence,e.g., a wild-type CSAPK sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in CSAPK genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control CSAPKnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner(1987) BiophysChem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions iswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA86:6230). Such allele specific oligonucleotides are hybridized to PCRamplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner et al. (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3'end of the 5' sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a CSAPK gene.

Furthermore, any cell type or tissue in which CSAPK is expressed may beutilized in the prognostic assays described herein.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs or compounds) on theexpression or activity of a CSAPK protein can be applied not only inbasic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase CSAPK gene expression, protein levels, or upregulateCSAPK activity, can be monitored in clinical trials of subjectsexhibiting decreased CSAPK gene expression, protein levels, ordownregulated CSAPK activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease CSAPK gene expression,protein levels, or downregulate CSAPK activity, can be monitored inclinical trials of subjects exhibiting increased CSAPK gene expression,protein levels, or upregulated CSAPK activity. In such clinical trials,the expression or activity of a CSAPK gene, and preferably, other genesthat have been implicated in a disorder can be used as a "read out" ormarkers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including CSAPK, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates CSAPK activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on a CSAPK associated disorder, for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe levels of expression of CSAPK and other genes implicated in theCSAPK associated disorder, respectively. The levels of gene expression(i.e., a gene expression pattern) can be quantified by Northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of protein produced, by one of the methods as describedherein, or by measuring the levels of activity of CSAPK or other genes.In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a CSAPK protein,mRNA, or genomic DNA in the pre-administration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the CSAPK protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the CSAPK protein, mRNA, or genomic DNA inthe pre-administration sample with the CSAPK protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of CSAPK to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of CSAPK to lower levels than detected, i.e. to decrease theeffectiveness of the agent. According to such an embodiment, CSAPKexpression or activity may be used as an indicator of the effectivenessof an agent, even in the absence of an observable phenotypic response.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant CSAPK expression oractivity. With regards to both prophylactic and therapeutic methods oftreatment, such treatments may be specifically tailored or modified,based on knowledge obtained from the field of pharmacogenomics."Pharmacogenomics", as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's "drugresponse phenotype", or "drug response genotype".) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the CSAPK molecules ofthe present invention or CSAPK modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant CSAPKexpression or activity, by administering to the subject a CSAPK or anagent which modulates CSAPK expression or at least one CSAPK activity.Subjects at risk for a disease which is caused or contributed to byaberrant CSAPK expression or activity can be identified by, for example,any or a combination of diagnostic or prognostic assays as describedherein. Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the CSAPK aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of CSAPK aberrancy, for example,a CSAPK, CSAPK agonist or CSAPK antagonist agent can be used fortreating the subject. The appropriate agent can be determined based onscreening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating CSAPKexpression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a CSAPK or agent that modulates one or more ofthe activities of CSAPK protein activity associated with the cell. Anagent that modulates CSAPK protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a CSAPK protein (e.g., a CSAPK phosphorylationsubstrate), a CSAPK antibody, a CSAPK agonist or antagonist, apeptidomimetic of a CSAPK agonist or antagonist, or other smallmolecule. In one embodiment, the agent stimulates one or more CSAPKactivities. Examples of such stimulatory agents include active CSAPKprotein and a nucleic acid molecule encoding CSAPK that has beenintroduced into the cell. In another embodiment, the agent inhibits oneor more CSAPK activites. Examples of such inhibitory agents includeantisense CSAPK nucleic acid molecules, anti-CSAPK antibodies, and CSAPKinhibitors. These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g, byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of a CSAPKprotein or nucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g.,upregulates or downregulates) CSAPK expression or activity. In anotherembodiment, the method involves administering a CSAPK protein or nucleicacid molecule as therapy to compensate for reduced or aberrant CSAPKexpression or activity.

Stimulation of CSAPK activity is desirable in situations in which CSAPKis abnormally downregulated and/or in which increased CSAPK activity islikely to have a beneficial effect. For example, stimulation of CSAPKactivity is desirable in situations in which a CSAPK is downregulatedand/or in which increased CSAPK activity is likely to have a beneficialeffect. Likewise, inhibition of CSAPK activity is desirable insituations in which CSAPK is abnormally upregulated and/or in whichdecreased CSAPK activity is likely to have a beneficial effect.

3. Pharmacogenomics

The CSAPK molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on CSAPKactivity (e.g., CSAPK gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) disorders (e.g, cardiovasculardisorders such as congestive heart failure) associated with aberrantCSAPK activity. In conjunction with such treatment, pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician may consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administer aCSAPK molecule or CSAPK modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with a CSAPK molecule or CSAPKmodulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofarans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as "a genome-wide association", relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a "bi-allelic" gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a "SNP" is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the "candidate gene approach", can beutilized to identify genes that predict a drug response. According tothis method, if a gene that encodes a drug target is known (e.g., aCSAPK protein or CSAPK receptor of the present invention), all commonvariants of that gene can be fairly easily identified in the populationand it can be determined if having one version of the gene versusanother is associated with a particular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the "gene expression profiling", can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a CSAPK moleculeor CSAPK modulator of the present invention) can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharnacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a CSAPK molecule orCSAPK modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Human CSAPKcDNA

In this example, the identification and characterization of the genesencoding human CSAPK-1, CSAPK-2, CSAPK-3, CSAPK-4, and CSAPK-5 (alsoreferred to as b004a10, b086g01, b007e05, b002d04, and b155a01,respectively) is described.

Isolation of the Human CSAPK cDNA

The invention is based, at least in part, on the discovery of five humangenes encoding members of the CSAPK family. The human CSAPK familymembers were isolated from cDNA libraries which were prepared fromtissue obtained from subjects suffering from congestive heart failure ofischemic and idiopathic origin. Briefly, a cardiac tissue sample wasobtained from a biopsy of a patient suffering from congestive heartfailure. mRNA was isolated from the cardiac tissue and a cDNA librarywas prepared therefrom using art known methods (described in, forexample, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor LaboratoryPress:1989). Positive clones were isolated following comparison tohomologs in public protein databases, including a comparison with knownkinases and/or examination of the sequence for protein motifs ofkinases.

The sequences of the positive clones were determined and found tocontain open reading frames. The nucleotide sequence encoding the humanCSAPK-1 protein is shown in FIG. 1 and is set forth as SEQ ID NO:1. Theprotein encoded by this nucleic acid comprises about 302 amino acids andhas the amino acid sequence shown in FIG. 1 and set forth as SEQ IDNO:2. The coding region (open reading frame) of SEQ ID NO:1 is set forthas SEQ ID NO:3. The clone comprising the entire coding region of humanCSAPK-1 was deposited with the American Type Culture Collection (ATCC®),10801 University Boulevard, Manassas, Va. 20110-2209, on Oct. 27, 1998,and assigned Accession Number 203308.

The nucleotide sequence encoding the human CSAPK-2 protein is shown inFIG. 2 and is set forth as SEQ ID NO:4. The protein encoded by thisnucleic acid comprises about 455 amino acids and has the amino acidsequence shown in FIG. 2 and set forth as SEQ ID NO:5. The coding region(open reading frame) of SEQ ID NO:4 is set forth as SEQ ID NO:6. Theclone comprising the entire coding region of human CSAPK-2 was depositedwith the American Type Culture Collection (ATCC®), 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Oct. 27, 1998 and assignedAccession Number 203306.

The nucleotide sequence encoding the human CSAPK-3 protein is shown inFIG. 3 and is set forth as SEQ ID NO:7. The protein encoded by thisnucleic acid comprises about 581 amino acids and has the amino acidsequence shown in FIG. 3 and set forth as SEQ ID NO:8. The coding region(open reading frame) of SEQ ID NO:7 is set forth as SEQ ID NO:9. Theclone comprising the entire coding region of human CSAPK-3 was depositedwith the American Type Culture Collection (ATCC®), 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Oct. 27, 1998 and assignedAccession Number 203309.

The nucleotide sequence encoding the human CSAPK-4 protein is shown inFIG. 4 and is set forth as SEQ ID NO:10. The protein encoded by thisnucleic acid comprises about 160 amino acids and has the amino acidsequence shown in FIG. 4 and set forth as SEQ ID NO:11. The codingregion (open reading frame) of SEQ ID NO:10 is set forth as SEQ IDNO:12. The clone comprising the entire coding region of human CSAPK-4was deposited with the American Type Culture Collection (ATCC®), 10801University Boulevard, Manassas, Va. 20110-2209, on Oct. 27, 1998 andassigned Accession Number 203307.

The nucleotide sequence encoding the human CSAPK-5 protein is shown inFIG. 5 and is set forth as SEQ ID NO:13. The protein encoded by thisnucleic acid comprises about 444 amino acids and has the amino acidsequence shown in FIG. 5 and set forth as SEQ ID NO:14. The codingregion (open reading frame) of SEQ ID NO:13 is set forth as SEQ IDNO:15.

Analysis of Human CSAPK Molecule

A BLASTN 1.4.9 search, using a score of 100 and a word length of 12(Altschul et al. (1990) J. Mol. Biol. 215:403) of the nucleotidesequence of human CSAPK-1 revealed that CSAPK-1 is similar to the humanprotein kinase HPK-1 coding sequence (Accession No. V23831). Thisnucleic acid molecule is approximately 70% identical to CSAPK-1, overnucleotides 388-1214.

CSAPK-2 is similar to the human MST mRNA for serine/threonine kinase(Accession No. Z48615). This nucleic acid molecule is approximately 54%identical to CSAPK-2, over nucleotides 482-805.

CSAPK-3 is similar to the human cDNA clone IMAGE:1257327 (Accession No.AA746653). This nucleic acid molecule is approximately 99% identical toCSAPK-3, over nucleotides 2068-2430.

CSAPK-4 is similar to the Mus musculus cDNA clone 902193 (Accession No.AA516800) This nucleic acid molecule is approximately 86% identical toCSAPK-4, over nucleotides 321-755.

CSAPK-5 is similar to the non-annotated human mRNA for KIAA0551 protein(Accession No. AB011123). This nucleic acid molecule is approximately100% identical to CSAPK-5, over nucleotides 1-1333.

Tissue Distribution of CSAPK mRNA

This Example describes the tissue distribution of CSAPK mRNA, asdetermined by Northern blot hybridizations.

Northern blot hybridizations with the various RNA samples were performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. The DNA probe was radioactively labeled with ³² P-dCTPusing the Prime-It kit (Stratagene, La Jolla, Calif.) according to theinstructions of the supplier. Filters containing human mRNA (MultiTissueNorthern I and MultiTissue Northern II from Clontech, Palo Alto, Calif.)were probed in ExpressHyb hybridization solution (Clontech) and washedat high stringency according to manufacturer's recommendations.

CSAPK-1 message was detected predominantly in the heart, skeletalmuscle, or the placenta. CSAPK-2 message was detected predominantly inmuscle, e.g., skeletal or cardiac muscle. CSAPK-3 message was detectedpredominantly in heart, skeletal muscle, brain, placenta, lung, liver,kidney, and pancreas. CSAPK-4 message was detected predominantly inskeletal muscle.

Example 2 Expression of recombinant CSAPK Protein in Bacterial Cells

In this example, CSAPK is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, CSAPK isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB 199. Expression of the GST-CSAPK fusion protein in PEB 199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB 199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 3 Expression of recombinant CSAPK Protein in COS Cells

To express the CSAPK gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire CSAPK protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3' end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

To construct the plasmid, the CSAPK DNA sequence is amplified by PCRusing two primers. The 5' primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the CSAPKcoding sequence starting from the initiation codon; the 3' end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the CSAPK coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the CSAPK gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5a, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the CSAPK-pcDNA/Amp plasmidDNA using the calcium phosphate or calcium chloride co-precipitationmethods, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Other suitable methods for transfecting host cells canbe found in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Theexpression of the CSAPK polypeptide is detected by radiolabelling (³⁵S-methionine or ³⁵ S-cysteine available from NEN, Boston, Mass., can beused) and immunoprecipitation (Harlow, E. and Lane, D.Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly,the cells are labelled for 8 hours with ³⁵ S-methionine (or ³⁵S-cysteine). The culture media are then collected and the cells arelysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS,0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the CSAPK coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the CSAPKpolypeptide is detected by radiolabelling and immunoprecipitation usinga CSAPK specific monoclonal antibody.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - <160> NUMBER OF SEQ ID NOS: 15                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 4137                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: 'n' at positions 28 - #72, 3597 and 3682 may be     any                                                                                  nucleic acid                                                            <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (297)..(1202)                                                  - - <400> SEQUENCE: 1                                                         - - tcgacccacg cgtccgggag gatcgggagt cgcgggagga tgggccgccg ct -             #aggctcgc     60                                                                 - - actccggacg cgcctcgcag tgcgcagggt gggtgccccg cgcctgcagc gt -            #ccgccggg    120                                                                 - - gcggcgcggc gggaggtggc cgacaggctc cgggcctcgc agcctcagcc cc -            #cggcccag    180                                                                 - - cgcgctttcc gacggcggcg ccgcgccgag ccacccgccc gcccaaggtc tc -            #tcgcgggc    240                                                                 - - gggagaacgg aaaactccca acttcctgag ttctaaagtt cctgttgctt ca - #gaca      atg    299                                                                                        - #                  - #                  - #             Met                                                                                               - #                  - #                  - #              1                                                                              - - gat gag caa tca caa gga atg caa ggg cca cc - #t gtt cct cag ttc caa          347                                                                       Asp Glu Gln Ser Gln Gly Met Gln Gly Pro Pr - #o Val Pro Gln Phe Gln                         5    - #              10    - #              15                  - - cca cag aag gcc tta cga ccg gat atg ggc ta - #t aat aca tta gcc aac          395                                                                       Pro Gln Lys Ala Leu Arg Pro Asp Met Gly Ty - #r Asn Thr Leu Ala Asn                    20         - #         25         - #         30                      - - ttt cga ata gaa aag aaa att ggt cgc gga ca - #a ttt agt gaa gtt tat          443                                                                       Phe Arg Ile Glu Lys Lys Ile Gly Arg Gly Gl - #n Phe Ser Glu Val Tyr                35             - #     40             - #     45                          - - aga gca gcc tgt ctc ttg gat gga gta cca gt - #a gct tta aaa aaa gtg          491                                                                       Arg Ala Ala Cys Leu Leu Asp Gly Val Pro Va - #l Ala Leu Lys Lys Val            50                 - # 55                 - # 60                 - # 65       - - cag ata ttt gat tta atg gat gcc aaa gca cg - #t gct gat tgc atc aaa          539                                                                       Gln Ile Phe Asp Leu Met Asp Ala Lys Ala Ar - #g Ala Asp Cys Ile Lys                            70 - #                 75 - #                 80              - - gaa ata gat ctt ctt aag caa ctc aac cat cc - #a aat gta ata aaa tat          587                                                                       Glu Ile Asp Leu Leu Lys Gln Leu Asn His Pr - #o Asn Val Ile Lys Tyr                        85     - #             90     - #             95                  - - tat gca tca ttc att gaa gat aat gaa cta aa - #c ata gtt ttg gaa cta          635                                                                       Tyr Ala Ser Phe Ile Glu Asp Asn Glu Leu As - #n Ile Val Leu Glu Leu                   100          - #       105          - #       110                      - - gca gat gct ggc gac cta tcc aga atg atc aa - #g cat ttt aag aag caa          683                                                                       Ala Asp Ala Gly Asp Leu Ser Arg Met Ile Ly - #s His Phe Lys Lys Gln               115              - #   120              - #   125                          - - aag agg cta att cct gaa aga act gtt tgg aa - #g tat ttt gtt cag ctt          731                                                                       Lys Arg Leu Ile Pro Glu Arg Thr Val Trp Ly - #s Tyr Phe Val Gln Leu           130                 1 - #35                 1 - #40                 1 -      #45                                                                              - - tgc agt gca ttg gaa cac atg cat tct cga ag - #a gtc atg cat aga        gat      779                                                                    Cys Ser Ala Leu Glu His Met His Ser Arg Ar - #g Val Met His Arg Asp                          150  - #               155  - #               160              - - ata aaa cca gct aat gtg ttc att aca gcc ac - #t ggg gtg gta aaa ctt          827                                                                       Ile Lys Pro Ala Asn Val Phe Ile Thr Ala Th - #r Gly Val Val Lys Leu                       165      - #           170      - #           175                  - - gga gat ctt ggg ctt ggc cgg ttt ttc agc tc - #a aaa acc aca gct gca          875                                                                       Gly Asp Leu Gly Leu Gly Arg Phe Phe Ser Se - #r Lys Thr Thr Ala Ala                   180          - #       185          - #       190                      - - cat tct tta gtt ggt acg cct tat tac atg tc - #t cca gag aga ata cat          923                                                                       His Ser Leu Val Gly Thr Pro Tyr Tyr Met Se - #r Pro Glu Arg Ile His               195              - #   200              - #   205                          - - gaa aat gga tac aac ttc aaa tct gac atc tg - #g tct ctt ggc tgt cta          971                                                                       Glu Asn Gly Tyr Asn Phe Lys Ser Asp Ile Tr - #p Ser Leu Gly Cys Leu           210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - cta tat gag atg gct gca tta caa agt cct tt - #c tat ggt gac aaa        atg     1019                                                                    Leu Tyr Glu Met Ala Ala Leu Gln Ser Pro Ph - #e Tyr Gly Asp Lys Met                          230  - #               235  - #               240              - - aat tta tac tca ctg tgt aag aag ata gaa ca - #g tgt gac tac cca cct         1067                                                                       Asn Leu Tyr Ser Leu Cys Lys Lys Ile Glu Gl - #n Cys Asp Tyr Pro Pro                       245      - #           250      - #           255                  - - ctt cct tca gat cac tat tca gaa gaa ctc cg - #a cag tta gtt aat atg         1115                                                                       Leu Pro Ser Asp His Tyr Ser Glu Glu Leu Ar - #g Gln Leu Val Asn Met                   260          - #       265          - #       270                      - - tgc atc aac cca gat cca gag aag cga cca ga - #c gtc acc tat gtt tat         1163                                                                       Cys Ile Asn Pro Asp Pro Glu Lys Arg Pro As - #p Val Thr Tyr Val Tyr               275              - #   280              - #   285                          - - gac gta gca aag agg atg cat gca tgc act gc - #a agc agc taaacatgca          1212                                                                       Asp Val Ala Lys Arg Met His Ala Cys Thr Al - #a Ser Ser                       290                 2 - #95                 3 - #00                            - - agatcatgaa gagtgtaacc aaagtaattg aaagtatttt gtgcaaagtc gt -             #acctsccc   1272                                                                 - - atttatgtct gggtgttaag attaatattt cagagctagt gtgctctgaa tc -            #cttaacca   1332                                                                 - - gttttcatat aagcttcatt ttgtaccagt cacctaaatc acctccttgc aa -            #cccccaaa   1392                                                                 - - tgactttgga ataactgaat tgcatgttag gagagaaaat gaaacatgat gg -            #ttttgaat   1452                                                                 - - ggctaaaggt ttatagaatt tcttacagtt ttctgctgat aaattgtgtt ta -            #gatagact   1512                                                                 - - gtcagtgcca aatattgaag gtgcagcttg gcacacatca gaatagactc at -            #acctgaga   1572                                                                 - - aaaagtatct gaacatgtga cttgtttctt ttttagtaat ttatggacat tg -            #agatgaac   1632                                                                 - - acaattgtga acttttgtga agattttatt tttaaacgtt tgaagtacta gt -            #tttagttc   1692                                                                 - - ttagcagagt agttttcaaa tatgattctt atgataaatg tagacacaaa ct -            #atttgaga   1752                                                                 - - aacatttaga actcttagct tatacattca aaatgtaact attaaatgtg aa -            #gatttggg   1812                                                                 - - gacaaaatgt gagtcagaca ctgaagagtt ttttgttttg ttttaatatt tt -            #tgatattc   1872                                                                 - - tctttgcatt gaaatggtat aaatgaatcc atttaaaaag tggttaagga tt -            #tgtttagc   1932                                                                 - - tggtgtgata ataattttta aagttgcaca ttgcccaagg ctttttttgt gt -            #gtttttat   1992                                                                 - - tgttgtttgt acatttgaaa aatattcttt gaataacctt gcagtactat at -            #ttcaagrt   2052                                                                 - - ttctttataa atttaagtgc attttaactc ataattgtac actataatat aa -            #gcctaagt   2112                                                                 - - ttttattcat aagttttatt gaagttctga tcggtcccct tcagaaattt tt -            #ttatatta   2172                                                                 - - ttcttcaagt tactttctta tttatattgt atgtgcattt tatccattaa tg -            #tttcatac   2232                                                                 - - tttctgagag tataataccc ttttaaaaga tatttggtat accaatactt tt -            #cctggatt   2292                                                                 - - gaaaactttt tttaaacttt ttaaaatttg ggccactctg tatgcatatg tt -            #tggtcttg   2352                                                                 - - ttaaagagga agaaaggatg tgtgttatac tgtacctgtg aatgttgata ca -            #gttacaat   2412                                                                 - - ttatttgaca aggttgtaat tctagaatat gcttaataaa atgaaaactg gc -            #catgacta   2472                                                                 - - cagccagaac tgttatgaga ttaacatttc tattgagaag cttttgagta aa -            #gtactgta   2532                                                                 - - tttgttcatg aagatgactg agatggtaac acttcgtgta gcttaaggaa at -            #gggcagaa   2592                                                                 - - tttcgtaaat gctgttgtgc agatgtgttt tccctgaatg ctttcgtatt ag -            #tggcgacc   2652                                                                 - - agtttctcac agaattgtga agcctgaagg ccaagaggaa gtcactgtta aa -            #ggactctg   2712                                                                 - - tgccatctta caaccttgga tgaattatcc tgccaacgtg aaaacctcat gt -            #tcaaagaa   2772                                                                 - - cacttccctt tagccgatgt aactgctggt tttgtttttc atatgtgttt tt -            #cttacact   2832                                                                 - - catttgaatg ctttcaagca tttgtaaact taaaaaaaan wawaaagggc aa -            #aaagtctg   2892                                                                 - - aacccttgtt ttctgaaatc taatcagtta tgtatggttt ctgaagggta at -            #tttatttt   2952                                                                 - - ggaataggta aagcgaaacc tgttttgtcw tgtttttcct gagggctaga tg -            #catttttt   3012                                                                 - - ttctcacact cttaatgact tttaacattt atactgagca tccatagata ta -            #ttcctaga   3072                                                                 - - agtatgagaa gaattattct tattgaccat taatgtcatg ttcattttaa tg -            #taatataa   3132                                                                 - - ttgagatgaa atgttctctg gttggaacag atactctctt tttttttctt gc -            #aatcttta   3192                                                                 - - agaatacata gatctaaaat tcattagctt gacccctcaa agtaactttt aa -            #gtaaagat   3252                                                                 - - taaagctttt cttctcagtg aatatatctg ctagaaggaa atagctggga ag -            #aatttaat   3312                                                                 - - gatcagggaa attcattatt tctatatgtg gaaacttttt gcttcgaata tt -            #gtatcttt   3372                                                                 - - ttaaatctaa atgttcatat ttttcctgaa gaaaccactg tgtaaaaatc aa -            #attttaat   3432                                                                 - - tttgaatgga ataatttcaa agaactatga agatgatttg aagctctaat tt -            #atatagtc   3492                                                                 - - acctataaaa tgttctttat atgtgttcat aagtaaattt tatattgatt aa -            #gttaaact   3552                                                                 - - tttgaattga tttgaggagc agtaaaatga aagctatatc tattnctaaa cc -            #ytatttag   3612                                                                 - - acattggkac cagttaccca ggtgaaaata kggagtaact ttgttttgta tg -            #gtaaggtt   3672                                                                 - - taggaatggn ggatgaaggg tatctctata taaataaagt gctcaacaat gt -            #gcaatgat   3732                                                                 - - tgtaaattta gtaagatatt acagccattt catgaatgct ttaccattca ac -            #atagtatc   3792                                                                 - - tattacaaaa cacctttctt gtatccatat acttcaggtg ttgctgttaa ca -            #tttactat   3852                                                                 - - gatatttatt ttaaccaaaa tgttactcac attaaatgtt tattctttaa aa -            #tgaatgta   3912                                                                 - - ttatgttttt aacccacaaa tgcatactta ccctgtgcct catatttcaa ta -            #gtactgta   3972                                                                 - - atatggacat cttttgtgaa atacttttat tttgttatgc tttaaatata ca -            #tacaaaaa   4032                                                                 - - gatttctgtt attagctttg aaaattgtat aatatcctaa tataacaaaa at -            #ataaaaat   4092                                                                 - - aaaaatgaat acagtaaaaa aaaaaaaaaa aaaaaaaaaa aaagg   - #                    4137                                                                        - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 302                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 2                                                         - - Met Asp Glu Gln Ser Gln Gly Met Gln Gly Pr - #o Pro Val Pro Gln Phe        1               5 - #                 10 - #                 15              - - Gln Pro Gln Lys Ala Leu Arg Pro Asp Met Gl - #y Tyr Asn Thr Leu Ala                   20     - #             25     - #             30                  - - Asn Phe Arg Ile Glu Lys Lys Ile Gly Arg Gl - #y Gln Phe Ser Glu Val               35         - #         40         - #         45                      - - Tyr Arg Ala Ala Cys Leu Leu Asp Gly Val Pr - #o Val Ala Leu Lys Lys           50             - #     55             - #     60                          - - Val Gln Ile Phe Asp Leu Met Asp Ala Lys Al - #a Arg Ala Asp Cys Ile       65                 - # 70                 - # 75                 - # 80       - - Lys Glu Ile Asp Leu Leu Lys Gln Leu Asn Hi - #s Pro Asn Val Ile Lys                       85 - #                 90 - #                 95              - - Tyr Tyr Ala Ser Phe Ile Glu Asp Asn Glu Le - #u Asn Ile Val Leu Glu                  100      - #           105      - #           110                  - - Leu Ala Asp Ala Gly Asp Leu Ser Arg Met Il - #e Lys His Phe Lys Lys              115          - #       120          - #       125                      - - Gln Lys Arg Leu Ile Pro Glu Arg Thr Val Tr - #p Lys Tyr Phe Val Gln          130              - #   135              - #   140                          - - Leu Cys Ser Ala Leu Glu His Met His Ser Ar - #g Arg Val Met His Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Ile Lys Pro Ala Asn Val Phe Ile Thr Al - #a Thr Gly Val Val        Lys                                                                                             165  - #               170  - #               175             - - Leu Gly Asp Leu Gly Leu Gly Arg Phe Phe Se - #r Ser Lys Thr Thr Ala                  180      - #           185      - #           190                  - - Ala His Ser Leu Val Gly Thr Pro Tyr Tyr Me - #t Ser Pro Glu Arg Ile              195          - #       200          - #       205                      - - His Glu Asn Gly Tyr Asn Phe Lys Ser Asp Il - #e Trp Ser Leu Gly Cys          210              - #   215              - #   220                          - - Leu Leu Tyr Glu Met Ala Ala Leu Gln Ser Pr - #o Phe Tyr Gly Asp Lys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Met Asn Leu Tyr Ser Leu Cys Lys Lys Ile Gl - #u Gln Cys Asp Tyr        Pro                                                                                             245  - #               250  - #               255             - - Pro Leu Pro Ser Asp His Tyr Ser Glu Glu Le - #u Arg Gln Leu Val Asn                  260      - #           265      - #           270                  - - Met Cys Ile Asn Pro Asp Pro Glu Lys Arg Pr - #o Asp Val Thr Tyr Val              275          - #       280          - #       285                      - - Tyr Asp Val Ala Lys Arg Met His Ala Cys Th - #r Ala Ser Ser                  290              - #   295              - #   300                          - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 906                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(906)                                                     - - <400> SEQUENCE: 3                                                         - - atg gat gag caa tca caa gga atg caa ggg cc - #a cct gtt cct cag ttc       48                                                                           Met Asp Glu Gln Ser Gln Gly Met Gln Gly Pr - #o Pro Val Pro Gln Phe             1               5 - #                 10 - #                 15              - - caa cca cag aag gcc tta cga ccg gat atg gg - #c tat aat aca tta gcc       96                                                                           Gln Pro Gln Lys Ala Leu Arg Pro Asp Met Gl - #y Tyr Asn Thr Leu Ala                        20     - #             25     - #             30                  - - aac ttt cga ata gaa aag aaa att ggt cgc gg - #a caa ttt agt gaa gtt       144                                                                          Asn Phe Arg Ile Glu Lys Lys Ile Gly Arg Gl - #y Gln Phe Ser Glu Val                    35         - #         40         - #         45                      - - tat aga gca gcc tgt ctc ttg gat gga gta cc - #a gta gct tta aaa aaa       192                                                                          Tyr Arg Ala Ala Cys Leu Leu Asp Gly Val Pr - #o Val Ala Leu Lys Lys                50             - #     55             - #     60                          - - gtg cag ata ttt gat tta atg gat gcc aaa gc - #a cgt gct gat tgc atc       240                                                                          Val Gln Ile Phe Asp Leu Met Asp Ala Lys Al - #a Arg Ala Asp Cys Ile            65                 - # 70                 - # 75                 - # 80       - - aaa gaa ata gat ctt ctt aag caa ctc aac ca - #t cca aat gta ata aaa       288                                                                          Lys Glu Ile Asp Leu Leu Lys Gln Leu Asn Hi - #s Pro Asn Val Ile Lys                            85 - #                 90 - #                 95              - - tat tat gca tca ttc att gaa gat aat gaa ct - #a aac ata gtt ttg gaa       336                                                                          Tyr Tyr Ala Ser Phe Ile Glu Asp Asn Glu Le - #u Asn Ile Val Leu Glu                       100      - #           105      - #           110                  - - cta gca gat gct ggc gac cta tcc aga atg at - #c aag cat ttt aag aag       384                                                                          Leu Ala Asp Ala Gly Asp Leu Ser Arg Met Il - #e Lys His Phe Lys Lys                   115          - #       120          - #       125                      - - caa aag agg cta att cct gaa aga act gtt tg - #g aag tat ttt gtt cag       432                                                                          Gln Lys Arg Leu Ile Pro Glu Arg Thr Val Tr - #p Lys Tyr Phe Val Gln               130              - #   135              - #   140                          - - ctt tgc agt gca ttg gaa cac atg cat tct cg - #a aga gtc atg cat aga       480                                                                          Leu Cys Ser Ala Leu Glu His Met His Ser Ar - #g Arg Val Met His Arg           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - gat ata aaa cca gct aat gtg ttc att aca gc - #c act ggg gtg gta        aaa   528                                                                       Asp Ile Lys Pro Ala Asn Val Phe Ile Thr Al - #a Thr Gly Val Val Lys                          165  - #               170  - #               175              - - ctt gga gat ctt ggg ctt ggc cgg ttt ttc ag - #c tca aaa acc aca gct       576                                                                          Leu Gly Asp Leu Gly Leu Gly Arg Phe Phe Se - #r Ser Lys Thr Thr Ala                       180      - #           185      - #           190                  - - gca cat tct tta gtt ggt acg cct tat tac at - #g tct cca gag aga ata       624                                                                          Ala His Ser Leu Val Gly Thr Pro Tyr Tyr Me - #t Ser Pro Glu Arg Ile                   195          - #       200          - #       205                      - - cat gaa aat gga tac aac ttc aaa tct gac at - #c tgg tct ctt ggc tgt       672                                                                          His Glu Asn Gly Tyr Asn Phe Lys Ser Asp Il - #e Trp Ser Leu Gly Cys               210              - #   215              - #   220                          - - cta cta tat gag atg gct gca tta caa agt cc - #t ttc tat ggt gac aaa       720                                                                          Leu Leu Tyr Glu Met Ala Ala Leu Gln Ser Pr - #o Phe Tyr Gly Asp Lys           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - atg aat tta tac tca ctg tgt aag aag ata ga - #a cag tgt gac tac        cca   768                                                                       Met Asn Leu Tyr Ser Leu Cys Lys Lys Ile Gl - #u Gln Cys Asp Tyr Pro                          245  - #               250  - #               255              - - cct ctt cct tca gat cac tat tca gaa gaa ct - #c cga cag tta gtt aat       816                                                                          Pro Leu Pro Ser Asp His Tyr Ser Glu Glu Le - #u Arg Gln Leu Val Asn                       260      - #           265      - #           270                  - - atg tgc atc aac cca gat cca gag aag cga cc - #a gac gtc acc tat gtt       864                                                                          Met Cys Ile Asn Pro Asp Pro Glu Lys Arg Pr - #o Asp Val Thr Tyr Val                   275          - #       280          - #       285                      - - tat gac gta gca aag agg atg cat gca tgc ac - #t gca agc agc               906                                                                          Tyr Asp Val Ala Lys Arg Met His Ala Cys Th - #r Ala Ser Ser                       290              - #   295              - #   300                          - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 2120                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (47)..(1411)                                                   - - <400> SEQUENCE: 4                                                         - - gtcgacccac gcgtccggtg gaagtataat actttgtcat tatgag atg - #tcg tct        55                                                                                              - #                  - #               Met Ser - #Ser                         - #                  - #                 1                   - - ctc ggt gcc tcc ttt gtg caa att aaa ttt ga - #t gac ttg cag ttt ttt       103                                                                          Leu Gly Ala Ser Phe Val Gln Ile Lys Phe As - #p Asp Leu Gln Phe Phe                 5            - #      10            - #      15                          - - gaa aac tgc ggt gga gga agt ttt ggg agt gt - #t tat cga gcc aaa tgg       151                                                                          Glu Asn Cys Gly Gly Gly Ser Phe Gly Ser Va - #l Tyr Arg Ala Lys Trp            20                 - # 25                 - # 30                 - # 35       - - ata tca cag gac aag gag gtg gct gta aag aa - #g ctc ctc aaa ata gag       199                                                                          Ile Ser Gln Asp Lys Glu Val Ala Val Lys Ly - #s Leu Leu Lys Ile Glu                            40 - #                 45 - #                 50              - - aaa gag gca gaa ata ctc agt gtc ctc agt ca - #c aga aac atc atc cag       247                                                                          Lys Glu Ala Glu Ile Leu Ser Val Leu Ser Hi - #s Arg Asn Ile Ile Gln                        55     - #             60     - #             65                  - - ttt tat gga gta att ctt gaa cct ccc aac ta - #t ggc att gtc aca gaa       295                                                                          Phe Tyr Gly Val Ile Leu Glu Pro Pro Asn Ty - #r Gly Ile Val Thr Glu                    70         - #         75         - #         80                      - - tat gct tct ctg gga tca ctc tat gat tac at - #t aac agt aac aga agt       343                                                                          Tyr Ala Ser Leu Gly Ser Leu Tyr Asp Tyr Il - #e Asn Ser Asn Arg Ser                85             - #     90             - #     95                          - - gag gag atg gat atg gat cac att atg acc tg - #g gcc act gat gta gcc       391                                                                          Glu Glu Met Asp Met Asp His Ile Met Thr Tr - #p Ala Thr Asp Val Ala           100                 1 - #05                 1 - #10                 1 -      #15                                                                              - - aaa gga atg cat tat tta cat atg gag gct cc - #t gtc aag gtg att        cac   439                                                                       Lys Gly Met His Tyr Leu His Met Glu Ala Pr - #o Val Lys Val Ile His                          120  - #               125  - #               130              - - aga gac ctc aag tca aga aac gtt gtt ata gc - #t gct gat gga gta ctg       487                                                                          Arg Asp Leu Lys Ser Arg Asn Val Val Ile Al - #a Ala Asp Gly Val Leu                       135      - #           140      - #           145                  - - aag atc tgt gac ttt ggt gcc tct cgg ttc ca - #t aac cat aca aca cac       535                                                                          Lys Ile Cys Asp Phe Gly Ala Ser Arg Phe Hi - #s Asn His Thr Thr His                   150          - #       155          - #       160                      - - atg tcc ttg gtt gga act ttc cca tgg atg gc - #t cca gaa gtt atc cag       583                                                                          Met Ser Leu Val Gly Thr Phe Pro Trp Met Al - #a Pro Glu Val Ile Gln               165              - #   170              - #   175                          - - agt ctc cct gtg tca gaa act tgt gac aca ta - #t tcc tat ggt gtg gtt       631                                                                          Ser Leu Pro Val Ser Glu Thr Cys Asp Thr Ty - #r Ser Tyr Gly Val Val           180                 1 - #85                 1 - #90                 1 -      #95                                                                              - - ctc tgg gag atg cta aca agg gag gtc ccc tt - #t aaa ggt ttg gaa        gga   679                                                                       Leu Trp Glu Met Leu Thr Arg Glu Val Pro Ph - #e Lys Gly Leu Glu Gly                          200  - #               205  - #               210              - - tta caa gta gct tgg ctt gta gtg gaa aaa aa - #c gag aga tta acc att       727                                                                          Leu Gln Val Ala Trp Leu Val Val Glu Lys As - #n Glu Arg Leu Thr Ile                       215      - #           220      - #           225                  - - cca agc agt tgc ccc aga agt ttt gct gaa ct - #g tta cat cag tgt tgg       775                                                                          Pro Ser Ser Cys Pro Arg Ser Phe Ala Glu Le - #u Leu His Gln Cys Trp                   230          - #       235          - #       240                      - - gaa gct gat gcc aag aaa cgg cca tca ttc aa - #g caa atc att tca atc       823                                                                          Glu Ala Asp Ala Lys Lys Arg Pro Ser Phe Ly - #s Gln Ile Ile Ser Ile               245              - #   250              - #   255                          - - ctg gag tcc atg tca aat gac acg agc ctt cc - #t gac aag tgt aac tca       871                                                                          Leu Glu Ser Met Ser Asn Asp Thr Ser Leu Pr - #o Asp Lys Cys Asn Ser           260                 2 - #65                 2 - #70                 2 -      #75                                                                              - - ttc cta cac aac aag gcg gag tgg agg tgc ga - #a att gag gca act        ctt   919                                                                       Phe Leu His Asn Lys Ala Glu Trp Arg Cys Gl - #u Ile Glu Ala Thr Leu                          280  - #               285  - #               290              - - gag agg cta aag aaa cta gag cgt gat ctc ag - #c ttt aag gag cag gag       967                                                                          Glu Arg Leu Lys Lys Leu Glu Arg Asp Leu Se - #r Phe Lys Glu Gln Glu                       295      - #           300      - #           305                  - - ctt aaa gaa cga gaa aga cgt tta aag atg tg - #g gag caa aag ctg aca       1015                                                                         Leu Lys Glu Arg Glu Arg Arg Leu Lys Met Tr - #p Glu Gln Lys Leu Thr                   310          - #       315          - #       320                      - - gag cag tcc aac acc ccg ctt ctc ttg cct ct - #t gct gca aga atg tct       1063                                                                         Glu Gln Ser Asn Thr Pro Leu Leu Leu Pro Le - #u Ala Ala Arg Met Ser               325              - #   330              - #   335                          - - gag gag tct tac ttt gaa tct aaa aca gag ga - #g tca aac agt gca gag       1111                                                                         Glu Glu Ser Tyr Phe Glu Ser Lys Thr Glu Gl - #u Ser Asn Ser Ala Glu           340                 3 - #45                 3 - #50                 3 -      #55                                                                              - - atg tca tgt cag atc aca gca aca agt aac gg - #g gag ggc cat ggc        atg   1159                                                                      Met Ser Cys Gln Ile Thr Ala Thr Ser Asn Gl - #y Glu Gly His Gly Met                          360  - #               365  - #               370              - - aac cca agt ctg cag gcc atg atg ctg atg gg - #c ttt ggg gat atc ttc       1207                                                                         Asn Pro Ser Leu Gln Ala Met Met Leu Met Gl - #y Phe Gly Asp Ile Phe                       375      - #           380      - #           385                  - - tca atg aac aaa gca gga gct gtg atg cat tc - #t ggg atg cag ata aac       1255                                                                         Ser Met Asn Lys Ala Gly Ala Val Met His Se - #r Gly Met Gln Ile Asn                   390          - #       395          - #       400                      - - atg caa gcc aag cag aat tct tcc aaa acc ac - #a tct aag aga agg ggg       1303                                                                         Met Gln Ala Lys Gln Asn Ser Ser Lys Thr Th - #r Ser Lys Arg Arg Gly               405              - #   410              - #   415                          - - aag aaa gtc aac atg gct ctg ggg ttc agt ga - #t ttt gac ttg tca gaa       1351                                                                         Lys Lys Val Asn Met Ala Leu Gly Phe Ser As - #p Phe Asp Leu Ser Glu           420                 4 - #25                 4 - #30                 4 -      #35                                                                              - - ggt gac gat gat gat gat gat gac ggt gag ga - #g gag gat aat gac        atg   1399                                                                      Gly Asp Asp Asp Asp Asp Asp Asp Gly Glu Gl - #u Glu Asp Asn Asp Met                          440  - #               445  - #               450              - - gat aat agt gaa tgaaagcaga aagcaaagta ataaaatcac aa - #atgtttgg           1451                                                                         Asp Asn Ser Glu                                                                           455                                                                - - aaaacacaaa agtaacttgt ttatctcagt ctgtacaaaa acagtaagga gg -             #cagaaagc 1511                                                                   - - caagcactgc atttttaggc caatcacatt tacatgaccg taatttctta tc -            #aattctac 1571                                                                   - - ttttattttt gcttacagaa aaacgggggg agaattaagc caaagaagta ta -            #tttatgaa 1631                                                                   - - tcagcaaatg tggtgcctga ttatagaaat ttgtgatcct atatacaata ta -            #ggactttt 1691                                                                   - - aaagttgtga cattctggct ttttctttta atgaatactt tttagtttgt at -            #ttgacttt 1751                                                                   - - atttccttta ttcaaatcat ttttaaaaac ttacattttg aacaaacact ct -            #taactcct 1811                                                                   - - aattgttctt tgacacgtag taattctgtg acatactttt tttttcttat ag -            #caatacac 1871                                                                   - - tgtaatatca gaaatggttg gcctgagcaa cctagtaaga cctcgtctct ac -            #taataatt 1931                                                                   - - aaaaaactag ctggcatggt agcacacacc tgtagtccca gatacttggg ag -            #gccaaggc 1991                                                                   - - aggaggattg cttgagacct agcaatcagt cagggctgca gtgagccatg at -            #ggcaccac 2051                                                                   - - tgcactctag cctgggcaag agaacaagat cctgtctcaa aaaacaaaaa aa -            #aaaaaaaa 2111                                                                   - - gggcggccg                - #                  - #                      - #     2120                                                                    - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 455                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 5                                                         - - Met Ser Ser Leu Gly Ala Ser Phe Val Gln Il - #e Lys Phe Asp Asp Leu        1               5 - #                 10 - #                 15              - - Gln Phe Phe Glu Asn Cys Gly Gly Gly Ser Ph - #e Gly Ser Val Tyr Arg                   20     - #             25     - #             30                  - - Ala Lys Trp Ile Ser Gln Asp Lys Glu Val Al - #a Val Lys Lys Leu Leu               35         - #         40         - #         45                      - - Lys Ile Glu Lys Glu Ala Glu Ile Leu Ser Va - #l Leu Ser His Arg Asn           50             - #     55             - #     60                          - - Ile Ile Gln Phe Tyr Gly Val Ile Leu Glu Pr - #o Pro Asn Tyr Gly Ile       65                 - # 70                 - # 75                 - # 80       - - Val Thr Glu Tyr Ala Ser Leu Gly Ser Leu Ty - #r Asp Tyr Ile Asn Ser                       85 - #                 90 - #                 95              - - Asn Arg Ser Glu Glu Met Asp Met Asp His Il - #e Met Thr Trp Ala Thr                  100      - #           105      - #           110                  - - Asp Val Ala Lys Gly Met His Tyr Leu His Me - #t Glu Ala Pro Val Lys              115          - #       120          - #       125                      - - Val Ile His Arg Asp Leu Lys Ser Arg Asn Va - #l Val Ile Ala Ala Asp          130              - #   135              - #   140                          - - Gly Val Leu Lys Ile Cys Asp Phe Gly Ala Se - #r Arg Phe His Asn His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Thr Thr His Met Ser Leu Val Gly Thr Phe Pr - #o Trp Met Ala Pro        Glu                                                                                             165  - #               170  - #               175             - - Val Ile Gln Ser Leu Pro Val Ser Glu Thr Cy - #s Asp Thr Tyr Ser Tyr                  180      - #           185      - #           190                  - - Gly Val Val Leu Trp Glu Met Leu Thr Arg Gl - #u Val Pro Phe Lys Gly              195          - #       200          - #       205                      - - Leu Glu Gly Leu Gln Val Ala Trp Leu Val Va - #l Glu Lys Asn Glu Arg          210              - #   215              - #   220                          - - Leu Thr Ile Pro Ser Ser Cys Pro Arg Ser Ph - #e Ala Glu Leu Leu His      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gln Cys Trp Glu Ala Asp Ala Lys Lys Arg Pr - #o Ser Phe Lys Gln        Ile                                                                                             245  - #               250  - #               255             - - Ile Ser Ile Leu Glu Ser Met Ser Asn Asp Th - #r Ser Leu Pro Asp Lys                  260      - #           265      - #           270                  - - Cys Asn Ser Phe Leu His Asn Lys Ala Glu Tr - #p Arg Cys Glu Ile Glu              275          - #       280          - #       285                      - - Ala Thr Leu Glu Arg Leu Lys Lys Leu Glu Ar - #g Asp Leu Ser Phe Lys          290              - #   295              - #   300                          - - Glu Gln Glu Leu Lys Glu Arg Glu Arg Arg Le - #u Lys Met Trp Glu Gln      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Leu Thr Glu Gln Ser Asn Thr Pro Leu Le - #u Leu Pro Leu Ala        Ala                                                                                             325  - #               330  - #               335             - - Arg Met Ser Glu Glu Ser Tyr Phe Glu Ser Ly - #s Thr Glu Glu Ser Asn                  340      - #           345      - #           350                  - - Ser Ala Glu Met Ser Cys Gln Ile Thr Ala Th - #r Ser Asn Gly Glu Gly              355          - #       360          - #       365                      - - His Gly Met Asn Pro Ser Leu Gln Ala Met Me - #t Leu Met Gly Phe Gly          370              - #   375              - #   380                          - - Asp Ile Phe Ser Met Asn Lys Ala Gly Ala Va - #l Met His Ser Gly Met      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gln Ile Asn Met Gln Ala Lys Gln Asn Ser Se - #r Lys Thr Thr Ser        Lys                                                                                             405  - #               410  - #               415             - - Arg Arg Gly Lys Lys Val Asn Met Ala Leu Gl - #y Phe Ser Asp Phe Asp                  420      - #           425      - #           430                  - - Leu Ser Glu Gly Asp Asp Asp Asp Asp Asp As - #p Gly Glu Glu Glu Asp              435          - #       440          - #       445                      - - Asn Asp Met Asp Asn Ser Glu                                                  450              - #   455                                                 - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 1365                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(1365)                                                    - - <400> SEQUENCE: 6                                                         - - atg tcg tct ctc ggt gcc tcc ttt gtg caa at - #t aaa ttt gat gac ttg       48                                                                           Met Ser Ser Leu Gly Ala Ser Phe Val Gln Il - #e Lys Phe Asp Asp Leu             1               5 - #                 10 - #                 15              - - cag ttt ttt gaa aac tgc ggt gga gga agt tt - #t ggg agt gtt tat cga       96                                                                           Gln Phe Phe Glu Asn Cys Gly Gly Gly Ser Ph - #e Gly Ser Val Tyr Arg                        20     - #             25     - #             30                  - - gcc aaa tgg ata tca cag gac aag gag gtg gc - #t gta aag aag ctc ctc       144                                                                          Ala Lys Trp Ile Ser Gln Asp Lys Glu Val Al - #a Val Lys Lys Leu Leu                    35         - #         40         - #         45                      - - aaa ata gag aaa gag gca gaa ata ctc agt gt - #c ctc agt cac aga aac       192                                                                          Lys Ile Glu Lys Glu Ala Glu Ile Leu Ser Va - #l Leu Ser His Arg Asn                50             - #     55             - #     60                          - - atc atc cag ttt tat gga gta att ctt gaa cc - #t ccc aac tat ggc att       240                                                                          Ile Ile Gln Phe Tyr Gly Val Ile Leu Glu Pr - #o Pro Asn Tyr Gly Ile            65                 - # 70                 - # 75                 - # 80       - - gtc aca gaa tat gct tct ctg gga tca ctc ta - #t gat tac att aac agt       288                                                                          Val Thr Glu Tyr Ala Ser Leu Gly Ser Leu Ty - #r Asp Tyr Ile Asn Ser                            85 - #                 90 - #                 95              - - aac aga agt gag gag atg gat atg gat cac at - #t atg acc tgg gcc act       336                                                                          Asn Arg Ser Glu Glu Met Asp Met Asp His Il - #e Met Thr Trp Ala Thr                       100      - #           105      - #           110                  - - gat gta gcc aaa gga atg cat tat tta cat at - #g gag gct cct gtc aag       384                                                                          Asp Val Ala Lys Gly Met His Tyr Leu His Me - #t Glu Ala Pro Val Lys                   115          - #       120          - #       125                      - - gtg att cac aga gac ctc aag tca aga aac gt - #t gtt ata gct gct gat       432                                                                          Val Ile His Arg Asp Leu Lys Ser Arg Asn Va - #l Val Ile Ala Ala Asp               130              - #   135              - #   140                          - - gga gta ctg aag atc tgt gac ttt ggt gcc tc - #t cgg ttc cat aac cat       480                                                                          Gly Val Leu Lys Ile Cys Asp Phe Gly Ala Se - #r Arg Phe His Asn His           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - aca aca cac atg tcc ttg gtt gga act ttc cc - #a tgg atg gct cca        gaa   528                                                                       Thr Thr His Met Ser Leu Val Gly Thr Phe Pr - #o Trp Met Ala Pro Glu                          165  - #               170  - #               175              - - gtt atc cag agt ctc cct gtg tca gaa act tg - #t gac aca tat tcc tat       576                                                                          Val Ile Gln Ser Leu Pro Val Ser Glu Thr Cy - #s Asp Thr Tyr Ser Tyr                       180      - #           185      - #           190                  - - ggt gtg gtt ctc tgg gag atg cta aca agg ga - #g gtc ccc ttt aaa ggt       624                                                                          Gly Val Val Leu Trp Glu Met Leu Thr Arg Gl - #u Val Pro Phe Lys Gly                   195          - #       200          - #       205                      - - ttg gaa gga tta caa gta gct tgg ctt gta gt - #g gaa aaa aac gag aga       672                                                                          Leu Glu Gly Leu Gln Val Ala Trp Leu Val Va - #l Glu Lys Asn Glu Arg               210              - #   215              - #   220                          - - tta acc att cca agc agt tgc ccc aga agt tt - #t gct gaa ctg tta cat       720                                                                          Leu Thr Ile Pro Ser Ser Cys Pro Arg Ser Ph - #e Ala Glu Leu Leu His           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - cag tgt tgg gaa gct gat gcc aag aaa cgg cc - #a tca ttc aag caa        atc   768                                                                       Gln Cys Trp Glu Ala Asp Ala Lys Lys Arg Pr - #o Ser Phe Lys Gln Ile                          245  - #               250  - #               255              - - att tca atc ctg gag tcc atg tca aat gac ac - #g agc ctt cct gac aag       816                                                                          Ile Ser Ile Leu Glu Ser Met Ser Asn Asp Th - #r Ser Leu Pro Asp Lys                       260      - #           265      - #           270                  - - tgt aac tca ttc cta cac aac aag gcg gag tg - #g agg tgc gaa att gag       864                                                                          Cys Asn Ser Phe Leu His Asn Lys Ala Glu Tr - #p Arg Cys Glu Ile Glu                   275          - #       280          - #       285                      - - gca act ctt gag agg cta aag aaa cta gag cg - #t gat ctc agc ttt aag       912                                                                          Ala Thr Leu Glu Arg Leu Lys Lys Leu Glu Ar - #g Asp Leu Ser Phe Lys               290              - #   295              - #   300                          - - gag cag gag ctt aaa gaa cga gaa aga cgt tt - #a aag atg tgg gag caa       960                                                                          Glu Gln Glu Leu Lys Glu Arg Glu Arg Arg Le - #u Lys Met Trp Glu Gln           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - aag ctg aca gag cag tcc aac acc ccg ctt ct - #c ttg cct ctt gct        gca   1008                                                                      Lys Leu Thr Glu Gln Ser Asn Thr Pro Leu Le - #u Leu Pro Leu Ala Ala                          325  - #               330  - #               335              - - aga atg tct gag gag tct tac ttt gaa tct aa - #a aca gag gag tca aac       1056                                                                         Arg Met Ser Glu Glu Ser Tyr Phe Glu Ser Ly - #s Thr Glu Glu Ser Asn                       340      - #           345      - #           350                  - - agt gca gag atg tca tgt cag atc aca gca ac - #a agt aac ggg gag ggc       1104                                                                         Ser Ala Glu Met Ser Cys Gln Ile Thr Ala Th - #r Ser Asn Gly Glu Gly                   355          - #       360          - #       365                      - - cat ggc atg aac cca agt ctg cag gcc atg at - #g ctg atg ggc ttt ggg       1152                                                                         His Gly Met Asn Pro Ser Leu Gln Ala Met Me - #t Leu Met Gly Phe Gly               370              - #   375              - #   380                          - - gat atc ttc tca atg aac aaa gca gga gct gt - #g atg cat tct ggg atg       1200                                                                         Asp Ile Phe Ser Met Asn Lys Ala Gly Ala Va - #l Met His Ser Gly Met           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - cag ata aac atg caa gcc aag cag aat tct tc - #c aaa acc aca tct        aag   1248                                                                      Gln Ile Asn Met Gln Ala Lys Gln Asn Ser Se - #r Lys Thr Thr Ser Lys                          405  - #               410  - #               415              - - aga agg ggg aag aaa gtc aac atg gct ctg gg - #g ttc agt gat ttt gac       1296                                                                         Arg Arg Gly Lys Lys Val Asn Met Ala Leu Gl - #y Phe Ser Asp Phe Asp                       420      - #           425      - #           430                  - - ttg tca gaa ggt gac gat gat gat gat gat ga - #c ggt gag gag gag gat       1344                                                                         Leu Ser Glu Gly Asp Asp Asp Asp Asp Asp As - #p Gly Glu Glu Glu Asp                   435          - #       440          - #       445                      - - aat gac atg gat aat agt gaa       - #                  - #                  1365                                                                       Asn Asp Met Asp Asn Ser Glu                                                       450              - #   455                                                 - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 2454                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (51)..(1793)                                                   - - <400> SEQUENCE: 7                                                         - - cggtggtggc ggcagcggcg gctgcggggg caccgggccg cggcgccacc at - #g gcg        56                                                                                             - #                  - #                  - # Met Ala                         - #                  - #                  - #   1            - - gtg cga cag gcg ctg ggc cgc ggc ctg cag ct - #g ggt cga gcg ctg ctg       104                                                                          Val Arg Gln Ala Leu Gly Arg Gly Leu Gln Le - #u Gly Arg Ala Leu Leu                     5        - #          10        - #          15                      - - ctg cgc ttc acg ggc aag ccc ggc cgg gcc ta - #c ggc ttg ggg cgg ccg       152                                                                          Leu Arg Phe Thr Gly Lys Pro Gly Arg Ala Ty - #r Gly Leu Gly Arg Pro                20             - #     25             - #     30                          - - ggc ccg gcg gcg ggc tgt gtc cgc ggg gag cg - #t cca ggc tgg gcc gca       200                                                                          Gly Pro Ala Ala Gly Cys Val Arg Gly Glu Ar - #g Pro Gly Trp Ala Ala            35                 - # 40                 - # 45                 - # 50       - - gga ccg ggc gcg gag cct cgc agg gtc ggg ct - #c ggg ctt cct aac cgt       248                                                                          Gly Pro Gly Ala Glu Pro Arg Arg Val Gly Le - #u Gly Leu Pro Asn Arg                            55 - #                 60 - #                 65              - - ctc cgc ttc ttc cgc cag tcg gtg gcc ggg ct - #g gcg gcg cgg ttg cag       296                                                                          Leu Arg Phe Phe Arg Gln Ser Val Ala Gly Le - #u Ala Ala Arg Leu Gln                        70     - #             75     - #             80                  - - cgg cag ttc gtg gtg cgg gcc tgg ggc tgc gc - #g ggc cct tgc ggc cgg       344                                                                          Arg Gln Phe Val Val Arg Ala Trp Gly Cys Al - #a Gly Pro Cys Gly Arg                    85         - #         90         - #         95                      - - gca gtc ttt ctg gcc ttc ggg cta ggg ctg gg - #c ctc atc gag gaa aaa       392                                                                          Ala Val Phe Leu Ala Phe Gly Leu Gly Leu Gl - #y Leu Ile Glu Glu Lys               100              - #   105              - #   110                          - - cag gcg gag agc cgg cgg gcg gtc tcg gcc tg - #t cag gag atc cag gca       440                                                                          Gln Ala Glu Ser Arg Arg Ala Val Ser Ala Cy - #s Gln Glu Ile Gln Ala           115                 1 - #20                 1 - #25                 1 -      #30                                                                              - - att ttt acc cag aaa agc aag ccg ggg cct ga - #c ccg ttg gac acg        aga   488                                                                       Ile Phe Thr Gln Lys Ser Lys Pro Gly Pro As - #p Pro Leu Asp Thr Arg                          135  - #               140  - #               145              - - cgc ttg cag ggc ttt cgg ctg gag gag tat ct - #g ata ggg cag tcc att       536                                                                          Arg Leu Gln Gly Phe Arg Leu Glu Glu Tyr Le - #u Ile Gly Gln Ser Ile                       150      - #           155      - #           160                  - - ggt aag ggc tgc agt gct gct gtg tat gaa gc - #c acc atg cct aca ttg       584                                                                          Gly Lys Gly Cys Ser Ala Ala Val Tyr Glu Al - #a Thr Met Pro Thr Leu                   165          - #       170          - #       175                      - - ccc cag aac ctg gag gtg aca aag agc acc gg - #g ttg ctt cca ggg aga       632                                                                          Pro Gln Asn Leu Glu Val Thr Lys Ser Thr Gl - #y Leu Leu Pro Gly Arg               180              - #   185              - #   190                          - - ggc cca ggt acc agt gca cca gga gaa ggg ca - #g gag cga gct ccg ggg       680                                                                          Gly Pro Gly Thr Ser Ala Pro Gly Glu Gly Gl - #n Glu Arg Ala Pro Gly           195                 2 - #00                 2 - #05                 2 -      #10                                                                              - - gcc cct gcc ttc ccc ttg gcc atc aag atg at - #g tgg aac atc tcg        gca   728                                                                       Ala Pro Ala Phe Pro Leu Ala Ile Lys Met Me - #t Trp Asn Ile Ser Ala                          215  - #               220  - #               225              - - ggt tcc tcc agc gaa gcc atc ttg aac aca at - #g agc cag gag ctg gtc       776                                                                          Gly Ser Ser Ser Glu Ala Ile Leu Asn Thr Me - #t Ser Gln Glu Leu Val                       230      - #           235      - #           240                  - - cca gcg agc cga gtg gcc ttg gct ggg gag ta - #t gga gca gtc act tac       824                                                                          Pro Ala Ser Arg Val Ala Leu Ala Gly Glu Ty - #r Gly Ala Val Thr Tyr                   245          - #       250          - #       255                      - - aga aaa tcc aag aga ggt ccc aag caa cta gc - #c cct cac ccc aac atc       872                                                                          Arg Lys Ser Lys Arg Gly Pro Lys Gln Leu Al - #a Pro His Pro Asn Ile               260              - #   265              - #   270                          - - atc cgg gtt ctc cgc gcc ttc acc tct tcc gt - #g ccg ctg ctg cca ggg       920                                                                          Ile Arg Val Leu Arg Ala Phe Thr Ser Ser Va - #l Pro Leu Leu Pro Gly           275                 2 - #80                 2 - #85                 2 -      #90                                                                              - - gcc ctg gtc gac tac cct gat gtg ctg ccc tc - #a cgc ctc cac cct        gaa   968                                                                       Ala Leu Val Asp Tyr Pro Asp Val Leu Pro Se - #r Arg Leu His Pro Glu                          295  - #               300  - #               305              - - ggc ctg ggc cat ggc cgg acg ctg ttc ctc gt - #t atg aag aac tat ccc       1016                                                                         Gly Leu Gly His Gly Arg Thr Leu Phe Leu Va - #l Met Lys Asn Tyr Pro                       310      - #           315      - #           320                  - - tgt acc ctg cgc cag tac ctt tgt gtg aac ac - #a ccc agc ccc cgc ctc       1064                                                                         Cys Thr Leu Arg Gln Tyr Leu Cys Val Asn Th - #r Pro Ser Pro Arg Leu                   325          - #       330          - #       335                      - - gcc gcc atg atg ctg ctg cag ctg ctg gaa gg - #c gtg gac cat ctg gtt       1112                                                                         Ala Ala Met Met Leu Leu Gln Leu Leu Glu Gl - #y Val Asp His Leu Val               340              - #   345              - #   350                          - - caa cag ggc atc gcg cac aga gac ctg aaa tc - #c gac aac atc ctt gtg       1160                                                                         Gln Gln Gly Ile Ala His Arg Asp Leu Lys Se - #r Asp Asn Ile Leu Val           355                 3 - #60                 3 - #65                 3 -      #70                                                                              - - gag ctg gac cca gac ggc tgc ccc tgg ctg gt - #g atc gca gat ttt        ggc   1208                                                                      Glu Leu Asp Pro Asp Gly Cys Pro Trp Leu Va - #l Ile Ala Asp Phe Gly                          375  - #               380  - #               385              - - tgc tgc ctg gct gat gag agc atc ggc ctg ca - #g ttg ccc ttc agc agc       1256                                                                         Cys Cys Leu Ala Asp Glu Ser Ile Gly Leu Gl - #n Leu Pro Phe Ser Ser                       390      - #           395      - #           400                  - - tgg tac gtg gat cgg ggc gga aac ggc tgt ct - #g atg gcc cca gag gtg       1304                                                                         Trp Tyr Val Asp Arg Gly Gly Asn Gly Cys Le - #u Met Ala Pro Glu Val                   405          - #       410          - #       415                      - - tcc acg gcc cgt cct ggc ccc agg gca gtg at - #t gac tac agc aag gct       1352                                                                         Ser Thr Ala Arg Pro Gly Pro Arg Ala Val Il - #e Asp Tyr Ser Lys Ala               420              - #   425              - #   430                          - - gat gcc tgg gca gtg gga gcc atc gcc tat ga - #a atc ttc ggg ctt gtc       1400                                                                         Asp Ala Trp Ala Val Gly Ala Ile Ala Tyr Gl - #u Ile Phe Gly Leu Val           435                 4 - #40                 4 - #45                 4 -      #50                                                                              - - aat ccc ttc tac ggc cag ggc aag gcc cac ct - #t gaa agc cgc agc        tac   1448                                                                      Asn Pro Phe Tyr Gly Gln Gly Lys Ala His Le - #u Glu Ser Arg Ser Tyr                          455  - #               460  - #               465              - - caa gag gct cag cta cct gca ctg ccc gag tc - #a gtg cct cca gac gtg       1496                                                                         Gln Glu Ala Gln Leu Pro Ala Leu Pro Glu Se - #r Val Pro Pro Asp Val                       470      - #           475      - #           480                  - - aga cag ttg gtg agg gca ctg ctc cag cga ga - #g gcc agc aag aga cca       1544                                                                         Arg Gln Leu Val Arg Ala Leu Leu Gln Arg Gl - #u Ala Ser Lys Arg Pro                   485          - #       490          - #       495                      - - tct gcc cga gta gcc gca aat gtg ctt cat ct - #a agc ctc tgg ggt gaa       1592                                                                         Ser Ala Arg Val Ala Ala Asn Val Leu His Le - #u Ser Leu Trp Gly Glu               500              - #   505              - #   510                          - - cat att cta gcc ctg aag aat ctg aag tta ga - #c aag atg gtt ggc tgg       1640                                                                         His Ile Leu Ala Leu Lys Asn Leu Lys Leu As - #p Lys Met Val Gly Trp           515                 5 - #20                 5 - #25                 5 -      #30                                                                              - - ctc ctc caa caa tcg gcc gcc act ttg ttg gc - #c aac agg ctc aca        gag   1688                                                                      Leu Leu Gln Gln Ser Ala Ala Thr Leu Leu Al - #a Asn Arg Leu Thr Glu                          535  - #               540  - #               545              - - aag tgt tgt gtg gaa aca aaa atg aag atg ct - #c ttt ctg gct aac ctg       1736                                                                         Lys Cys Cys Val Glu Thr Lys Met Lys Met Le - #u Phe Leu Ala Asn Leu                       550      - #           555      - #           560                  - - gag tgt gaa acg ctc tgc cag gca gcc ctc ct - #c ctc tgc tca tgg agg       1784                                                                         Glu Cys Glu Thr Leu Cys Gln Ala Ala Leu Le - #u Leu Cys Ser Trp Arg                   565          - #       570          - #       575                      - - gca gcc ctg tgatgtccct gcatggagct ggtgaattac taaaagaac - #t               1833                                                                         Ala Ala Leu                                                                       580                                                                        - - tggcatcctc tgtgtcgtga tggtctgtga atggtgaggg tgggagtcag ga -             #gacaagac 1893                                                                   - - agcgcagaga gggctggtta gccggaaaag gcctcgggct tggcaaatgg aa -            #gaacttga 1953                                                                   - - gtgagagttc agtctgcagt cctctgctca cagacatctg aaaagtgaat gg -            #ccaagctg 2013                                                                   - - gtctagtaga tgaggctgga ctgaggaggg gtaggcctgc atccacagag ag -            #gatccagg 2073                                                                   - - ccaaggcact ggctgtcagt ggcagagttt ggctgtgacc tttgccccta ac -            #acgaggaa 2133                                                                   - - ctcgtttgaa gggggcagcg tagcatgtct gatttgccac ctggatgaag gc -            #agacatca 2193                                                                   - - acatgggtca gcacgttcag ttacgggagt gggaaattac atgaggcctg gg -            #cctctgcg 2253                                                                   - - ttcccaagct gtgcgttctg gaccagctac tgaattatta atctcactta gc -            #gaaagtga 2313                                                                   - - cggatgagca gtaagtaagt aagtgtgggg atttaaactt gagggtttcc ct -            #cctgacta 2373                                                                   - - gcctctctta caggaattgt gaaatattaa atgcaaattt acaactgcaa aa -            #aaaaaaaa 2433                                                                   - - aaaaaaaaaa aagggcggcc c           - #                  - #                  2454                                                                       - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 581                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 8                                                         - - Met Ala Val Arg Gln Ala Leu Gly Arg Gly Le - #u Gln Leu Gly Arg Ala        1               5 - #                 10 - #                 15              - - Leu Leu Leu Arg Phe Thr Gly Lys Pro Gly Ar - #g Ala Tyr Gly Leu Gly                   20     - #             25     - #             30                  - - Arg Pro Gly Pro Ala Ala Gly Cys Val Arg Gl - #y Glu Arg Pro Gly Trp               35         - #         40         - #         45                      - - Ala Ala Gly Pro Gly Ala Glu Pro Arg Arg Va - #l Gly Leu Gly Leu Pro           50             - #     55             - #     60                          - - Asn Arg Leu Arg Phe Phe Arg Gln Ser Val Al - #a Gly Leu Ala Ala Arg       65                 - # 70                 - # 75                 - # 80       - - Leu Gln Arg Gln Phe Val Val Arg Ala Trp Gl - #y Cys Ala Gly Pro Cys                       85 - #                 90 - #                 95              - - Gly Arg Ala Val Phe Leu Ala Phe Gly Leu Gl - #y Leu Gly Leu Ile Glu                  100      - #           105      - #           110                  - - Glu Lys Gln Ala Glu Ser Arg Arg Ala Val Se - #r Ala Cys Gln Glu Ile              115          - #       120          - #       125                      - - Gln Ala Ile Phe Thr Gln Lys Ser Lys Pro Gl - #y Pro Asp Pro Leu Asp          130              - #   135              - #   140                          - - Thr Arg Arg Leu Gln Gly Phe Arg Leu Glu Gl - #u Tyr Leu Ile Gly Gln      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ser Ile Gly Lys Gly Cys Ser Ala Ala Val Ty - #r Glu Ala Thr Met        Pro                                                                                             165  - #               170  - #               175             - - Thr Leu Pro Gln Asn Leu Glu Val Thr Lys Se - #r Thr Gly Leu Leu Pro                  180      - #           185      - #           190                  - - Gly Arg Gly Pro Gly Thr Ser Ala Pro Gly Gl - #u Gly Gln Glu Arg Ala              195          - #       200          - #       205                      - - Pro Gly Ala Pro Ala Phe Pro Leu Ala Ile Ly - #s Met Met Trp Asn Ile          210              - #   215              - #   220                          - - Ser Ala Gly Ser Ser Ser Glu Ala Ile Leu As - #n Thr Met Ser Gln Glu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Val Pro Ala Ser Arg Val Ala Leu Ala Gl - #y Glu Tyr Gly Ala        Val                                                                                             245  - #               250  - #               255             - - Thr Tyr Arg Lys Ser Lys Arg Gly Pro Lys Gl - #n Leu Ala Pro His Pro                  260      - #           265      - #           270                  - - Asn Ile Ile Arg Val Leu Arg Ala Phe Thr Se - #r Ser Val Pro Leu Leu              275          - #       280          - #       285                      - - Pro Gly Ala Leu Val Asp Tyr Pro Asp Val Le - #u Pro Ser Arg Leu His          290              - #   295              - #   300                          - - Pro Glu Gly Leu Gly His Gly Arg Thr Leu Ph - #e Leu Val Met Lys Asn      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Tyr Pro Cys Thr Leu Arg Gln Tyr Leu Cys Va - #l Asn Thr Pro Ser        Pro                                                                                             325  - #               330  - #               335             - - Arg Leu Ala Ala Met Met Leu Leu Gln Leu Le - #u Glu Gly Val Asp His                  340      - #           345      - #           350                  - - Leu Val Gln Gln Gly Ile Ala His Arg Asp Le - #u Lys Ser Asp Asn Ile              355          - #       360          - #       365                      - - Leu Val Glu Leu Asp Pro Asp Gly Cys Pro Tr - #p Leu Val Ile Ala Asp          370              - #   375              - #   380                          - - Phe Gly Cys Cys Leu Ala Asp Glu Ser Ile Gl - #y Leu Gln Leu Pro Phe      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ser Ser Trp Tyr Val Asp Arg Gly Gly Asn Gl - #y Cys Leu Met Ala        Pro                                                                                             405  - #               410  - #               415             - - Glu Val Ser Thr Ala Arg Pro Gly Pro Arg Al - #a Val Ile Asp Tyr Ser                  420      - #           425      - #           430                  - - Lys Ala Asp Ala Trp Ala Val Gly Ala Ile Al - #a Tyr Glu Ile Phe Gly              435          - #       440          - #       445                      - - Leu Val Asn Pro Phe Tyr Gly Gln Gly Lys Al - #a His Leu Glu Ser Arg          450              - #   455              - #   460                          - - Ser Tyr Gln Glu Ala Gln Leu Pro Ala Leu Pr - #o Glu Ser Val Pro Pro      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Asp Val Arg Gln Leu Val Arg Ala Leu Leu Gl - #n Arg Glu Ala Ser        Lys                                                                                             485  - #               490  - #               495             - - Arg Pro Ser Ala Arg Val Ala Ala Asn Val Le - #u His Leu Ser Leu Trp                  500      - #           505      - #           510                  - - Gly Glu His Ile Leu Ala Leu Lys Asn Leu Ly - #s Leu Asp Lys Met Val              515          - #       520          - #       525                      - - Gly Trp Leu Leu Gln Gln Ser Ala Ala Thr Le - #u Leu Ala Asn Arg Leu          530              - #   535              - #   540                          - - Thr Glu Lys Cys Cys Val Glu Thr Lys Met Ly - #s Met Leu Phe Leu Ala      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Asn Leu Glu Cys Glu Thr Leu Cys Gln Ala Al - #a Leu Leu Leu Cys        Ser                                                                                             565  - #               570  - #               575             - - Trp Arg Ala Ala Leu                                                                  580                                                                - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 1743                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(1743)                                                    - - <400> SEQUENCE: 9                                                         - - atg gcg gtg cga cag gcg ctg ggc cgc ggc ct - #g cag ctg ggt cga gcg       48                                                                           Met Ala Val Arg Gln Ala Leu Gly Arg Gly Le - #u Gln Leu Gly Arg Ala             1               5 - #                 10 - #                 15              - - ctg ctg ctg cgc ttc acg ggc aag ccc ggc cg - #g gcc tac ggc ttg ggg       96                                                                           Leu Leu Leu Arg Phe Thr Gly Lys Pro Gly Ar - #g Ala Tyr Gly Leu Gly                        20     - #             25     - #             30                  - - cgg ccg ggc ccg gcg gcg ggc tgt gtc cgc gg - #g gag cgt cca ggc tgg       144                                                                          Arg Pro Gly Pro Ala Ala Gly Cys Val Arg Gl - #y Glu Arg Pro Gly Trp                    35         - #         40         - #         45                      - - gcc gca gga ccg ggc gcg gag cct cgc agg gt - #c ggg ctc ggg ctt cct       192                                                                          Ala Ala Gly Pro Gly Ala Glu Pro Arg Arg Va - #l Gly Leu Gly Leu Pro                50             - #     55             - #     60                          - - aac cgt ctc cgc ttc ttc cgc cag tcg gtg gc - #c ggg ctg gcg gcg cgg       240                                                                          Asn Arg Leu Arg Phe Phe Arg Gln Ser Val Al - #a Gly Leu Ala Ala Arg            65                 - # 70                 - # 75                 - # 80       - - ttg cag cgg cag ttc gtg gtg cgg gcc tgg gg - #c tgc gcg ggc cct tgc       288                                                                          Leu Gln Arg Gln Phe Val Val Arg Ala Trp Gl - #y Cys Ala Gly Pro Cys                            85 - #                 90 - #                 95              - - ggc cgg gca gtc ttt ctg gcc ttc ggg cta gg - #g ctg ggc ctc atc gag       336                                                                          Gly Arg Ala Val Phe Leu Ala Phe Gly Leu Gl - #y Leu Gly Leu Ile Glu                       100      - #           105      - #           110                  - - gaa aaa cag gcg gag agc cgg cgg gcg gtc tc - #g gcc tgt cag gag atc       384                                                                          Glu Lys Gln Ala Glu Ser Arg Arg Ala Val Se - #r Ala Cys Gln Glu Ile                   115          - #       120          - #       125                      - - cag gca att ttt acc cag aaa agc aag ccg gg - #g cct gac ccg ttg gac       432                                                                          Gln Ala Ile Phe Thr Gln Lys Ser Lys Pro Gl - #y Pro Asp Pro Leu Asp               130              - #   135              - #   140                          - - acg aga cgc ttg cag ggc ttt cgg ctg gag ga - #g tat ctg ata ggg cag       480                                                                          Thr Arg Arg Leu Gln Gly Phe Arg Leu Glu Gl - #u Tyr Leu Ile Gly Gln           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - tcc att ggt aag ggc tgc agt gct gct gtg ta - #t gaa gcc acc atg        cct   528                                                                       Ser Ile Gly Lys Gly Cys Ser Ala Ala Val Ty - #r Glu Ala Thr Met Pro                          165  - #               170  - #               175              - - aca ttg ccc cag aac ctg gag gtg aca aag ag - #c acc ggg ttg ctt cca       576                                                                          Thr Leu Pro Gln Asn Leu Glu Val Thr Lys Se - #r Thr Gly Leu Leu Pro                       180      - #           185      - #           190                  - - ggg aga ggc cca ggt acc agt gca cca gga ga - #a ggg cag gag cga gct       624                                                                          Gly Arg Gly Pro Gly Thr Ser Ala Pro Gly Gl - #u Gly Gln Glu Arg Ala                   195          - #       200          - #       205                      - - ccg ggg gcc cct gcc ttc ccc ttg gcc atc aa - #g atg atg tgg aac atc       672                                                                          Pro Gly Ala Pro Ala Phe Pro Leu Ala Ile Ly - #s Met Met Trp Asn Ile               210              - #   215              - #   220                          - - tcg gca ggt tcc tcc agc gaa gcc atc ttg aa - #c aca atg agc cag gag       720                                                                          Ser Ala Gly Ser Ser Ser Glu Ala Ile Leu As - #n Thr Met Ser Gln Glu           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - ctg gtc cca gcg agc cga gtg gcc ttg gct gg - #g gag tat gga gca        gtc   768                                                                       Leu Val Pro Ala Ser Arg Val Ala Leu Ala Gl - #y Glu Tyr Gly Ala Val                          245  - #               250  - #               255              - - act tac aga aaa tcc aag aga ggt ccc aag ca - #a cta gcc cct cac ccc       816                                                                          Thr Tyr Arg Lys Ser Lys Arg Gly Pro Lys Gl - #n Leu Ala Pro His Pro                       260      - #           265      - #           270                  - - aac atc atc cgg gtt ctc cgc gcc ttc acc tc - #t tcc gtg ccg ctg ctg       864                                                                          Asn Ile Ile Arg Val Leu Arg Ala Phe Thr Se - #r Ser Val Pro Leu Leu                   275          - #       280          - #       285                      - - cca ggg gcc ctg gtc gac tac cct gat gtg ct - #g ccc tca cgc ctc cac       912                                                                          Pro Gly Ala Leu Val Asp Tyr Pro Asp Val Le - #u Pro Ser Arg Leu His               290              - #   295              - #   300                          - - cct gaa ggc ctg ggc cat ggc cgg acg ctg tt - #c ctc gtt atg aag aac       960                                                                          Pro Glu Gly Leu Gly His Gly Arg Thr Leu Ph - #e Leu Val Met Lys Asn           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - tat ccc tgt acc ctg cgc cag tac ctt tgt gt - #g aac aca ccc agc        ccc   1008                                                                      Tyr Pro Cys Thr Leu Arg Gln Tyr Leu Cys Va - #l Asn Thr Pro Ser Pro                          325  - #               330  - #               335              - - cgc ctc gcc gcc atg atg ctg ctg cag ctg ct - #g gaa ggc gtg gac cat       1056                                                                         Arg Leu Ala Ala Met Met Leu Leu Gln Leu Le - #u Glu Gly Val Asp His                       340      - #           345      - #           350                  - - ctg gtt caa cag ggc atc gcg cac aga gac ct - #g aaa tcc gac aac atc       1104                                                                         Leu Val Gln Gln Gly Ile Ala His Arg Asp Le - #u Lys Ser Asp Asn Ile                   355          - #       360          - #       365                      - - ctt gtg gag ctg gac cca gac ggc tgc ccc tg - #g ctg gtg atc gca gat       1152                                                                         Leu Val Glu Leu Asp Pro Asp Gly Cys Pro Tr - #p Leu Val Ile Ala Asp               370              - #   375              - #   380                          - - ttt ggc tgc tgc ctg gct gat gag agc atc gg - #c ctg cag ttg ccc ttc       1200                                                                         Phe Gly Cys Cys Leu Ala Asp Glu Ser Ile Gl - #y Leu Gln Leu Pro Phe           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - agc agc tgg tac gtg gat cgg ggc gga aac gg - #c tgt ctg atg gcc        cca   1248                                                                      Ser Ser Trp Tyr Val Asp Arg Gly Gly Asn Gl - #y Cys Leu Met Ala Pro                          405  - #               410  - #               415              - - gag gtg tcc acg gcc cgt cct ggc ccc agg gc - #a gtg att gac tac agc       1296                                                                         Glu Val Ser Thr Ala Arg Pro Gly Pro Arg Al - #a Val Ile Asp Tyr Ser                       420      - #           425      - #           430                  - - aag gct gat gcc tgg gca gtg gga gcc atc gc - #c tat gaa atc ttc ggg       1344                                                                         Lys Ala Asp Ala Trp Ala Val Gly Ala Ile Al - #a Tyr Glu Ile Phe Gly                   435          - #       440          - #       445                      - - ctt gtc aat ccc ttc tac ggc cag ggc aag gc - #c cac ctt gaa agc cgc       1392                                                                         Leu Val Asn Pro Phe Tyr Gly Gln Gly Lys Al - #a His Leu Glu Ser Arg               450              - #   455              - #   460                          - - agc tac caa gag gct cag cta cct gca ctg cc - #c gag tca gtg cct cca       1440                                                                         Ser Tyr Gln Glu Ala Gln Leu Pro Ala Leu Pr - #o Glu Ser Val Pro Pro           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - gac gtg aga cag ttg gtg agg gca ctg ctc ca - #g cga gag gcc agc        aag   1488                                                                      Asp Val Arg Gln Leu Val Arg Ala Leu Leu Gl - #n Arg Glu Ala Ser Lys                          485  - #               490  - #               495              - - aga cca tct gcc cga gta gcc gca aat gtg ct - #t cat cta agc ctc tgg       1536                                                                         Arg Pro Ser Ala Arg Val Ala Ala Asn Val Le - #u His Leu Ser Leu Trp                       500      - #           505      - #           510                  - - ggt gaa cat att cta gcc ctg aag aat ctg aa - #g tta gac aag atg gtt       1584                                                                         Gly Glu His Ile Leu Ala Leu Lys Asn Leu Ly - #s Leu Asp Lys Met Val                   515          - #       520          - #       525                      - - ggc tgg ctc ctc caa caa tcg gcc gcc act tt - #g ttg gcc aac agg ctc       1632                                                                         Gly Trp Leu Leu Gln Gln Ser Ala Ala Thr Le - #u Leu Ala Asn Arg Leu               530              - #   535              - #   540                          - - aca gag aag tgt tgt gtg gaa aca aaa atg aa - #g atg ctc ttt ctg gct       1680                                                                         Thr Glu Lys Cys Cys Val Glu Thr Lys Met Ly - #s Met Leu Phe Leu Ala           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - aac ctg gag tgt gaa acg ctc tgc cag gca gc - #c ctc ctc ctc tgc        tca   1728                                                                      Asn Leu Glu Cys Glu Thr Leu Cys Gln Ala Al - #a Leu Leu Leu Cys Ser                          565  - #               570  - #               575              - - tgg agg gca gcc ctg           - #                  - #                      - #1743                                                                    Trp Arg Ala Ala Leu                                                                       580                                                                - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 1864                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (275)..(754)                                                   - - <400> SEQUENCE: 10                                                        - - gtcgacccac gcggtccgcc cacgcgttcc ggagacatgt ctctgtgttt ct -             #ctcccctc 60                                                                     - - cgcttttgag tccgttgaag acacaatttc tctctgtcgg gtgcttagga gg -            #agctccat 120                                                                    - - gaacatgtat tgaattggac ttagctgaac aggctgctgg ttggctgccc ag -            #agggggca 180                                                                    - - ggctgtgttg ctgggagcct tccagctccc tgcagcagtc atggggcagg gt -            #tccccgag 240                                                                    - - tccgtaatcc ccatttccac ctactttccc ttag tta ttt gat t - #cc ctg tct      gtc 295                                                                                           - #                  - #  Leu Phe Asp Ser Leu Ser Val                        - #                  - #    1              - # 5             - - gta ctc agc tta agt gga gca tcc cct ttc ct - #g gga gac acg aag cag       343                                                                          Val Leu Ser Leu Ser Gly Ala Ser Pro Phe Le - #u Gly Asp Thr Lys Gln                    10         - #         15         - #         20                      - - gaa aca ctg gca aat atc aca gca gtg agt ta - #c gac ttt gat gag gaa       391                                                                          Glu Thr Leu Ala Asn Ile Thr Ala Val Ser Ty - #r Asp Phe Asp Glu Glu                25             - #     30             - #     35                          - - ttc ttc agc cag acg agc gag ctg gcc aag ga - #c ttt att cgg aag ctt       439                                                                          Phe Phe Ser Gln Thr Ser Glu Leu Ala Lys As - #p Phe Ile Arg Lys Leu            40                 - # 45                 - # 50                 - # 55       - - ctg gtt aaa gag acc cgg aaa cgg ctc aca at - #c caa gag gct ctc aga       487                                                                          Leu Val Lys Glu Thr Arg Lys Arg Leu Thr Il - #e Gln Glu Ala Leu Arg                            60 - #                 65 - #                 70              - - cac ccc tgg atc acg ccg gtg gac aac cag ca - #a gcc atg gtg cgc agg       535                                                                          His Pro Trp Ile Thr Pro Val Asp Asn Gln Gl - #n Ala Met Val Arg Arg                        75     - #             80     - #             85                  - - gag tct gtg gtc aat ctg gag aac ttc agg aa - #g cag tat gtc cgc agg       583                                                                          Glu Ser Val Val Asn Leu Glu Asn Phe Arg Ly - #s Gln Tyr Val Arg Arg                    90         - #         95         - #        100                      - - cgg tgg aag ctt tcc ttc agc atc gtg tcc ct - #g tgc aac cac ctc acc       631                                                                          Arg Trp Lys Leu Ser Phe Ser Ile Val Ser Le - #u Cys Asn His Leu Thr               105              - #   110              - #   115                          - - cgc tcg ctg atg aag aag gtg cac ctg agg cc - #g gat gag gac ctg agg       679                                                                          Arg Ser Leu Met Lys Lys Val His Leu Arg Pr - #o Asp Glu Asp Leu Arg           120                 1 - #25                 1 - #30                 1 -      #35                                                                              - - aac tgt gag agt gac act gag gag gac atc gc - #c agg agg aaa gcc        ctc   727                                                                       Asn Cys Glu Ser Asp Thr Glu Glu Asp Ile Al - #a Arg Arg Lys Ala Leu                          140  - #               145  - #               150              - - cac cca cgg agg agg agc agc acc tcc taactggcc - #t gacctgcagt             774                                                                          His Pro Arg Arg Arg Ser Ser Thr Ser                                                       155      - #           160                                         - - ggccgccagg gaggtctggg cccagcgggg ctcccttctg tgcagacttt tg -             #gacccagc 834                                                                    - - tcagcaccag cacccgggcg tcctgagcac tttgcaagag agatgggccc aa -            #ggaattca 894                                                                    - - gaagagcttg caggcaagcc aggagaccct gggagctgtg gctgtcttct gt -            #ggaggagg 954                                                                    - - ctccagcatt cccaaagctc ttaattctcc ataaaatggg ctttcctctg tc -            #tgccatcc 1014                                                                   - - tcagagtctg gggtgggagt gtggacttag gaaaacaata taaaggacat cc -            #tcatcatc 1074                                                                   - - acggggtgaa ggtcagacta aggcagcctt cttcacaggc tgagggggtt ca -            #gaaccagc 1134                                                                   - - ctggccaaaa attacaccag agagacagag tcctccccat tgggaacagg gt -            #gattgagg 1194                                                                   - - aaagtgaacc ttgggtgtga gggaccaatc ctgtgacctc ccagaaccat gg -            #aagccagg 1254                                                                   - - acgtcaggct gaccaacacc tcagaccttc tgaagcagcc cattgctggc cc -            #gccatgtt 1314                                                                   - - gtaattttgc tcatttttat taaacttctg gtttacctga tgcttggctt ct -            #tttagggc 1374                                                                   - - tacccccatc tcatttcctt tagcccgtgt gcctgtaact ctgagggggg gc -            #acccagtg 1434                                                                   - - gggtgctgag tgggcagaat ctcagaaggt cctcctgaac cgtccgcgca gg -            #cctgcagt 1494                                                                   - - gggcctgcct cctccttgct tccctaacag gaaggtgtcc agttcaagag aa -            #cccaccca 1554                                                                   - - gagactggga gtggtggctc acgcctataa tccctgcgct ttggcagtcc ga -            #ggcagggg 1614                                                                   - - aattgcttga actcaggagt tggagaccag cctgggcaac atggcaaaac gc -            #agtctgta 1674                                                                   - - caaaaaatac aaaaaattag ccaggtgtag gggtaggcac ctggcatccc ag -            #ctactcca 1734                                                                   - - ggggctgagg tgacagcatt gcttaagccc agaaggtcga ggctgcagtg ag -            #ctgagatc 1794                                                                   - - acgccactgc actccagtct gggtgacaga gagagaccat atccaaaaaa aa -            #aaaaaaaa 1854                                                                   - - gggcggccgc                - #                  - #                      - #    1864                                                                    - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 160                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 11                                                        - - Leu Phe Asp Ser Leu Ser Val Val Leu Ser Le - #u Ser Gly Ala Ser Pro        1               5 - #                 10 - #                 15              - - Phe Leu Gly Asp Thr Lys Gln Glu Thr Leu Al - #a Asn Ile Thr Ala Val                   20     - #             25     - #             30                  - - Ser Tyr Asp Phe Asp Glu Glu Phe Phe Ser Gl - #n Thr Ser Glu Leu Ala               35         - #         40         - #         45                      - - Lys Asp Phe Ile Arg Lys Leu Leu Val Lys Gl - #u Thr Arg Lys Arg Leu           50             - #     55             - #     60                          - - Thr Ile Gln Glu Ala Leu Arg His Pro Trp Il - #e Thr Pro Val Asp Asn       65                 - # 70                 - # 75                 - # 80       - - Gln Gln Ala Met Val Arg Arg Glu Ser Val Va - #l Asn Leu Glu Asn Phe                       85 - #                 90 - #                 95              - - Arg Lys Gln Tyr Val Arg Arg Arg Trp Lys Le - #u Ser Phe Ser Ile Val                  100      - #           105      - #           110                  - - Ser Leu Cys Asn His Leu Thr Arg Ser Leu Me - #t Lys Lys Val His Leu              115          - #       120          - #       125                      - - Arg Pro Asp Glu Asp Leu Arg Asn Cys Glu Se - #r Asp Thr Glu Glu Asp          130              - #   135              - #   140                          - - Ile Ala Arg Arg Lys Ala Leu His Pro Arg Ar - #g Arg Ser Ser Thr Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 480                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(480)                                                     - - <400> SEQUENCE: 12                                                        - - tta ttt gat tcc ctg tct gtc gta ctc agc tt - #a agt gga gca tcc        cct   48                                                                        Leu Phe Asp Ser Leu Ser Val Val Leu Ser Le - #u Ser Gly Ala Ser Pro            1               5 - #                 10 - #                 15              - - ttc ctg gga gac acg aag cag gaa aca ctg gc - #a aat atc aca gca gtg       96                                                                           Phe Leu Gly Asp Thr Lys Gln Glu Thr Leu Al - #a Asn Ile Thr Ala Val                        20     - #             25     - #             30                  - - agt tac gac ttt gat gag gaa ttc ttc agc ca - #g acg agc gag ctg gcc       144                                                                          Ser Tyr Asp Phe Asp Glu Glu Phe Phe Ser Gl - #n Thr Ser Glu Leu Ala                    35         - #         40         - #         45                      - - aag gac ttt att cgg aag ctt ctg gtt aaa ga - #g acc cgg aaa cgg ctc       192                                                                          Lys Asp Phe Ile Arg Lys Leu Leu Val Lys Gl - #u Thr Arg Lys Arg Leu                50             - #     55             - #     60                          - - aca atc caa gag gct ctc aga cac ccc tgg at - #c acg ccg gtg gac aac       240                                                                          Thr Ile Gln Glu Ala Leu Arg His Pro Trp Il - #e Thr Pro Val Asp Asn            65                 - # 70                 - # 75                 - # 80       - - cag caa gcc atg gtg cgc agg gag tct gtg gt - #c aat ctg gag aac ttc       288                                                                          Gln Gln Ala Met Val Arg Arg Glu Ser Val Va - #l Asn Leu Glu Asn Phe                            85 - #                 90 - #                 95              - - agg aag cag tat gtc cgc agg cgg tgg aag ct - #t tcc ttc agc atc gtg       336                                                                          Arg Lys Gln Tyr Val Arg Arg Arg Trp Lys Le - #u Ser Phe Ser Ile Val                       100      - #           105      - #           110                  - - tcc ctg tgc aac cac ctc acc cgc tcg ctg at - #g aag aag gtg cac ctg       384                                                                          Ser Leu Cys Asn His Leu Thr Arg Ser Leu Me - #t Lys Lys Val His Leu                   115          - #       120          - #       125                      - - agg ccg gat gag gac ctg agg aac tgt gag ag - #t gac act gag gag gac       432                                                                          Arg Pro Asp Glu Asp Leu Arg Asn Cys Glu Se - #r Asp Thr Glu Glu Asp               130              - #   135              - #   140                          - - atc gcc agg agg aaa gcc ctc cac cca cgg ag - #g agg agc agc acc tcc       480                                                                          Ile Ala Arg Arg Lys Ala Leu His Pro Arg Ar - #g Arg Ser Ser Thr Ser           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 1333                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (2)..(1333)                                                    - - <400> SEQUENCE: 13                                                        - - g acg gca tta gcc aaa gaa cta aga gaa ctc - #cgg att gaa gaa aca       aac 49                                                                            Thr Ala Leu Ala Lys Glu Leu Arg Glu L - #eu Arg Ile Glu Glu Thr Asn            1              - # 5                 - # 10                 - # 15         - - cgc cca atg aag aag gtg act gat tac tcc tc - #c tcc agt gag gag tca       97                                                                           Arg Pro Met Lys Lys Val Thr Asp Tyr Ser Se - #r Ser Ser Glu Glu Ser                        20     - #             25     - #             30                  - - gaa agt agc gag gaa gag gag gaa gat gga ga - #g agc gag acc cat gat       145                                                                          Glu Ser Ser Glu Glu Glu Glu Glu Asp Gly Gl - #u Ser Glu Thr His Asp                    35         - #         40         - #         45                      - - ggg aca gtg gct gtc agc gac ata ccc aga ct - #g ata cca aca gga gct       193                                                                          Gly Thr Val Ala Val Ser Asp Ile Pro Arg Le - #u Ile Pro Thr Gly Ala                50             - #     55             - #     60                          - - cca ggc agc aac gag cag tac aat gtg gga at - #g gtg ggg acg cat ggg       241                                                                          Pro Gly Ser Asn Glu Gln Tyr Asn Val Gly Me - #t Val Gly Thr His Gly            65                 - # 70                 - # 75                 - # 80       - - ctg gag acc tct cat gcg gac agt ttc agc gg - #c agt att tca aga gaa       289                                                                          Leu Glu Thr Ser His Ala Asp Ser Phe Ser Gl - #y Ser Ile Ser Arg Glu                            85 - #                 90 - #                 95              - - gga acc ttg atg att aga gag acg tct gga ga - #g aag aag cga tct ggc       337                                                                          Gly Thr Leu Met Ile Arg Glu Thr Ser Gly Gl - #u Lys Lys Arg Ser Gly                       100      - #           105      - #           110                  - - cac agt gac agc aat ggc ttt gct ggc cac at - #c aac ctc cct gac ctg       385                                                                          His Ser Asp Ser Asn Gly Phe Ala Gly His Il - #e Asn Leu Pro Asp Leu                   115          - #       120          - #       125                      - - gtg cag cag agc cat tct cca gct gga acc cc - #g act gag gga ctg ggg       433                                                                          Val Gln Gln Ser His Ser Pro Ala Gly Thr Pr - #o Thr Glu Gly Leu Gly               130              - #   135              - #   140                          - - cgc gtc tca acc cat tcc cag gag atg gac tc - #t ggg act gaa tat ggc       481                                                                          Arg Val Ser Thr His Ser Gln Glu Met Asp Se - #r Gly Thr Glu Tyr Gly           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - atg ggg agc agc acc aaa gcc tcc ttc acc cc - #c ttt gtg gac ccc        aga   529                                                                       Met Gly Ser Ser Thr Lys Ala Ser Phe Thr Pr - #o Phe Val Asp Pro Arg                          165  - #               170  - #               175              - - gta tac cag acg tct ccc act gat gaa gat ga - #a gag gat gag gaa tca       577                                                                          Val Tyr Gln Thr Ser Pro Thr Asp Glu Asp Gl - #u Glu Asp Glu Glu Ser                       180      - #           185      - #           190                  - - tca gcc gca gct ctg ttt act agc gaa ctt ct - #t agg caa gaa cag gcc       625                                                                          Ser Ala Ala Ala Leu Phe Thr Ser Glu Leu Le - #u Arg Gln Glu Gln Ala                   195          - #       200          - #       205                      - - aaa ctc aat gaa gca aga aag att tcg gtg gt - #a aat gta aac cca acc       673                                                                          Lys Leu Asn Glu Ala Arg Lys Ile Ser Val Va - #l Asn Val Asn Pro Thr               210              - #   215              - #   220                          - - aac att cgg cct cat agc gac aca cca gaa at - #c aga aaa tac aag aaa       721                                                                          Asn Ile Arg Pro His Ser Asp Thr Pro Glu Il - #e Arg Lys Tyr Lys Lys           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - cga ttc aac tca gaa ata ctt tgt gca gct ct - #g tgg ggt gta aac        ctt   769                                                                       Arg Phe Asn Ser Glu Ile Leu Cys Ala Ala Le - #u Trp Gly Val Asn Leu                          245  - #               250  - #               255              - - ctg gtg ggg act gaa aat ggc ctg atg ctt tt - #g gac cga agt ggg caa       817                                                                          Leu Val Gly Thr Glu Asn Gly Leu Met Leu Le - #u Asp Arg Ser Gly Gln                       260      - #           265      - #           270                  - - ggc aaa gtc tat aat ctg atc aac cgg agg cg - #a ttt cag cag atg gat       865                                                                          Gly Lys Val Tyr Asn Leu Ile Asn Arg Arg Ar - #g Phe Gln Gln Met Asp                   275          - #       280          - #       285                      - - gtg cta gag gga ctg aat gtc ctt gtg aca at - #t tca gga aag aag aat       913                                                                          Val Leu Glu Gly Leu Asn Val Leu Val Thr Il - #e Ser Gly Lys Lys Asn               290              - #   295              - #   300                          - - aag cta cga gtt tac tat ctt tca tgg tta ag - #a aac aga ata cta cat       961                                                                          Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Ar - #g Asn Arg Ile Leu His           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - aat gac cca gaa gta gaa aag aaa caa ggc tg - #g atc act gtt ggg        gac   1009                                                                      Asn Asp Pro Glu Val Glu Lys Lys Gln Gly Tr - #p Ile Thr Val Gly Asp                          325  - #               330  - #               335              - - ttg gaa ggc tgt ata cat tat aaa gtt gtt aa - #a tat gaa agg atc aaa       1057                                                                         Leu Glu Gly Cys Ile His Tyr Lys Val Val Ly - #s Tyr Glu Arg Ile Lys                       340      - #           345      - #           350                  - - ttt ttg gtg att gcc tta aag aat gct gtg ga - #a ata tat gct tgg gct       1105                                                                         Phe Leu Val Ile Ala Leu Lys Asn Ala Val Gl - #u Ile Tyr Ala Trp Ala                   355          - #       360          - #       365                      - - cct aaa ccg tat cat aaa ttc atg gca ttt aa - #g tct ttt gca gat ctc       1153                                                                         Pro Lys Pro Tyr His Lys Phe Met Ala Phe Ly - #s Ser Phe Ala Asp Leu               370              - #   375              - #   380                          - - cag cac aag cct ctg cta gtt gat ctc acg gt - #a gaa gaa ggt caa aga       1201                                                                         Gln His Lys Pro Leu Leu Val Asp Leu Thr Va - #l Glu Glu Gly Gln Arg           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - tta aag gtt att ttt ggt tca cac act ggt tt - #c cat gta att gat        gtt   1249                                                                      Leu Lys Val Ile Phe Gly Ser His Thr Gly Ph - #e His Val Ile Asp Val                          405  - #               410  - #               415              - - gat tca gga aac tct tat gat atc tac ata cc - #a tct cat att cag ggc       1297                                                                         Asp Ser Gly Asn Ser Tyr Asp Ile Tyr Ile Pr - #o Ser His Ile Gln Gly                       420      - #           425      - #           430                  - - aat atc act cct cat gct att gtc atc ttg cc - #t aaa                     - #   1333                                                                      Asn Ile Thr Pro His Ala Ile Val Ile Leu Pr - #o Lys                                   435          - #       440                                             - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 444                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 14                                                        - - Thr Ala Leu Ala Lys Glu Leu Arg Glu Leu Ar - #g Ile Glu Glu Thr        Asn                                                                               1               5 - #                 10 - #                 15             - - Arg Pro Met Lys Lys Val Thr Asp Tyr Ser Se - #r Ser Ser Glu Glu Ser                   20     - #             25     - #             30                  - - Glu Ser Ser Glu Glu Glu Glu Glu Asp Gly Gl - #u Ser Glu Thr His Asp               35         - #         40         - #         45                      - - Gly Thr Val Ala Val Ser Asp Ile Pro Arg Le - #u Ile Pro Thr Gly Ala           50             - #     55             - #     60                          - - Pro Gly Ser Asn Glu Gln Tyr Asn Val Gly Me - #t Val Gly Thr His Gly       65                 - # 70                 - # 75                 - # 80       - - Leu Glu Thr Ser His Ala Asp Ser Phe Ser Gl - #y Ser Ile Ser Arg Glu                       85 - #                 90 - #                 95              - - Gly Thr Leu Met Ile Arg Glu Thr Ser Gly Gl - #u Lys Lys Arg Ser Gly                  100      - #           105      - #           110                  - - His Ser Asp Ser Asn Gly Phe Ala Gly His Il - #e Asn Leu Pro Asp Leu              115          - #       120          - #       125                      - - Val Gln Gln Ser His Ser Pro Ala Gly Thr Pr - #o Thr Glu Gly Leu Gly          130              - #   135              - #   140                          - - Arg Val Ser Thr His Ser Gln Glu Met Asp Se - #r Gly Thr Glu Tyr Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Met Gly Ser Ser Thr Lys Ala Ser Phe Thr Pr - #o Phe Val Asp Pro        Arg                                                                                             165  - #               170  - #               175             - - Val Tyr Gln Thr Ser Pro Thr Asp Glu Asp Gl - #u Glu Asp Glu Glu Ser                  180      - #           185      - #           190                  - - Ser Ala Ala Ala Leu Phe Thr Ser Glu Leu Le - #u Arg Gln Glu Gln Ala              195          - #       200          - #       205                      - - Lys Leu Asn Glu Ala Arg Lys Ile Ser Val Va - #l Asn Val Asn Pro Thr          210              - #   215              - #   220                          - - Asn Ile Arg Pro His Ser Asp Thr Pro Glu Il - #e Arg Lys Tyr Lys Lys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Arg Phe Asn Ser Glu Ile Leu Cys Ala Ala Le - #u Trp Gly Val Asn        Leu                                                                                             245  - #               250  - #               255             - - Leu Val Gly Thr Glu Asn Gly Leu Met Leu Le - #u Asp Arg Ser Gly Gln                  260      - #           265      - #           270                  - - Gly Lys Val Tyr Asn Leu Ile Asn Arg Arg Ar - #g Phe Gln Gln Met Asp              275          - #       280          - #       285                      - - Val Leu Glu Gly Leu Asn Val Leu Val Thr Il - #e Ser Gly Lys Lys Asn          290              - #   295              - #   300                          - - Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Ar - #g Asn Arg Ile Leu His      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asn Asp Pro Glu Val Glu Lys Lys Gln Gly Tr - #p Ile Thr Val Gly        Asp                                                                                             325  - #               330  - #               335             - - Leu Glu Gly Cys Ile His Tyr Lys Val Val Ly - #s Tyr Glu Arg Ile Lys                  340      - #           345      - #           350                  - - Phe Leu Val Ile Ala Leu Lys Asn Ala Val Gl - #u Ile Tyr Ala Trp Ala              355          - #       360          - #       365                      - - Pro Lys Pro Tyr His Lys Phe Met Ala Phe Ly - #s Ser Phe Ala Asp Leu          370              - #   375              - #   380                          - - Gln His Lys Pro Leu Leu Val Asp Leu Thr Va - #l Glu Glu Gly Gln Arg      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Leu Lys Val Ile Phe Gly Ser His Thr Gly Ph - #e His Val Ile Asp        Val                                                                                             405  - #               410  - #               415             - - Asp Ser Gly Asn Ser Tyr Asp Ile Tyr Ile Pr - #o Ser His Ile Gln Gly                  420      - #           425      - #           430                  - - Asn Ile Thr Pro His Ala Ile Val Ile Leu Pr - #o Lys                              435          - #       440                                             - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 1332                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(1332)                                                    - - <400> SEQUENCE: 15                                                        - - acg gca tta gcc aaa gaa cta aga gaa ctc cg - #g att gaa gaa aca aac       48                                                                           Thr Ala Leu Ala Lys Glu Leu Arg Glu Leu Ar - #g Ile Glu Glu Thr Asn             1               5 - #                 10 - #                 15              - - cgc cca atg aag aag gtg act gat tac tcc tc - #c tcc agt gag gag tca       96                                                                           Arg Pro Met Lys Lys Val Thr Asp Tyr Ser Se - #r Ser Ser Glu Glu Ser                        20     - #             25     - #             30                  - - gaa agt agc gag gaa gag gag gaa gat gga ga - #g agc gag acc cat gat       144                                                                          Glu Ser Ser Glu Glu Glu Glu Glu Asp Gly Gl - #u Ser Glu Thr His Asp                    35         - #         40         - #         45                      - - ggg aca gtg gct gtc agc gac ata ccc aga ct - #g ata cca aca gga gct       192                                                                          Gly Thr Val Ala Val Ser Asp Ile Pro Arg Le - #u Ile Pro Thr Gly Ala                50             - #     55             - #     60                          - - cca ggc agc aac gag cag tac aat gtg gga at - #g gtg ggg acg cat ggg       240                                                                          Pro Gly Ser Asn Glu Gln Tyr Asn Val Gly Me - #t Val Gly Thr His Gly            65                 - # 70                 - # 75                 - # 80       - - ctg gag acc tct cat gcg gac agt ttc agc gg - #c agt att tca aga gaa       288                                                                          Leu Glu Thr Ser His Ala Asp Ser Phe Ser Gl - #y Ser Ile Ser Arg Glu                            85 - #                 90 - #                 95              - - gga acc ttg atg att aga gag acg tct gga ga - #g aag aag cga tct ggc       336                                                                          Gly Thr Leu Met Ile Arg Glu Thr Ser Gly Gl - #u Lys Lys Arg Ser Gly                       100      - #           105      - #           110                  - - cac agt gac agc aat ggc ttt gct ggc cac at - #c aac ctc cct gac ctg       384                                                                          His Ser Asp Ser Asn Gly Phe Ala Gly His Il - #e Asn Leu Pro Asp Leu                   115          - #       120          - #       125                      - - gtg cag cag agc cat tct cca gct gga acc cc - #g act gag gga ctg ggg       432                                                                          Val Gln Gln Ser His Ser Pro Ala Gly Thr Pr - #o Thr Glu Gly Leu Gly               130              - #   135              - #   140                          - - cgc gtc tca acc cat tcc cag gag atg gac tc - #t ggg act gaa tat ggc       480                                                                          Arg Val Ser Thr His Ser Gln Glu Met Asp Se - #r Gly Thr Glu Tyr Gly           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - atg ggg agc agc acc aaa gcc tcc ttc acc cc - #c ttt gtg gac ccc        aga   528                                                                       Met Gly Ser Ser Thr Lys Ala Ser Phe Thr Pr - #o Phe Val Asp Pro Arg                          165  - #               170  - #               175              - - gta tac cag acg tct ccc act gat gaa gat ga - #a gag gat gag gaa tca       576                                                                          Val Tyr Gln Thr Ser Pro Thr Asp Glu Asp Gl - #u Glu Asp Glu Glu Ser                       180      - #           185      - #           190                  - - tca gcc gca gct ctg ttt act agc gaa ctt ct - #t agg caa gaa cag gcc       624                                                                          Ser Ala Ala Ala Leu Phe Thr Ser Glu Leu Le - #u Arg Gln Glu Gln Ala                   195          - #       200          - #       205                      - - aaa ctc aat gaa gca aga aag att tcg gtg gt - #a aat gta aac cca acc       672                                                                          Lys Leu Asn Glu Ala Arg Lys Ile Ser Val Va - #l Asn Val Asn Pro Thr               210              - #   215              - #   220                          - - aac att cgg cct cat agc gac aca cca gaa at - #c aga aaa tac aag aaa       720                                                                          Asn Ile Arg Pro His Ser Asp Thr Pro Glu Il - #e Arg Lys Tyr Lys Lys           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - cga ttc aac tca gaa ata ctt tgt gca gct ct - #g tgg ggt gta aac        ctt   768                                                                       Arg Phe Asn Ser Glu Ile Leu Cys Ala Ala Le - #u Trp Gly Val Asn Leu                          245  - #               250  - #               255              - - ctg gtg ggg act gaa aat ggc ctg atg ctt tt - #g gac cga agt ggg caa       816                                                                          Leu Val Gly Thr Glu Asn Gly Leu Met Leu Le - #u Asp Arg Ser Gly Gln                       260      - #           265      - #           270                  - - ggc aaa gtc tat aat ctg atc aac cgg agg cg - #a ttt cag cag atg gat       864                                                                          Gly Lys Val Tyr Asn Leu Ile Asn Arg Arg Ar - #g Phe Gln Gln Met Asp                   275          - #       280          - #       285                      - - gtg cta gag gga ctg aat gtc ctt gtg aca at - #t tca gga aag aag aat       912                                                                          Val Leu Glu Gly Leu Asn Val Leu Val Thr Il - #e Ser Gly Lys Lys Asn               290              - #   295              - #   300                          - - aag cta cga gtt tac tat ctt tca tgg tta ag - #a aac aga ata cta cat       960                                                                          Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Ar - #g Asn Arg Ile Leu His           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - aat gac cca gaa gta gaa aag aaa caa ggc tg - #g atc act gtt ggg        gac   1008                                                                      Asn Asp Pro Glu Val Glu Lys Lys Gln Gly Tr - #p Ile Thr Val Gly Asp                          325  - #               330  - #               335              - - ttg gaa ggc tgt ata cat tat aaa gtt gtt aa - #a tat gaa agg atc aaa       1056                                                                         Leu Glu Gly Cys Ile His Tyr Lys Val Val Ly - #s Tyr Glu Arg Ile Lys                       340      - #           345      - #           350                  - - ttt ttg gtg att gcc tta aag aat gct gtg ga - #a ata tat gct tgg gct       1104                                                                         Phe Leu Val Ile Ala Leu Lys Asn Ala Val Gl - #u Ile Tyr Ala Trp Ala                   355          - #       360          - #       365                      - - cct aaa ccg tat cat aaa ttc atg gca ttt aa - #g tct ttt gca gat ctc       1152                                                                         Pro Lys Pro Tyr His Lys Phe Met Ala Phe Ly - #s Ser Phe Ala Asp Leu               370              - #   375              - #   380                          - - cag cac aag cct ctg cta gtt gat ctc acg gt - #a gaa gaa ggt caa aga       1200                                                                         Gln His Lys Pro Leu Leu Val Asp Leu Thr Va - #l Glu Glu Gly Gln Arg           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - tta aag gtt att ttt ggt tca cac act ggt tt - #c cat gta att gat        gtt   1248                                                                      Leu Lys Val Ile Phe Gly Ser His Thr Gly Ph - #e His Val Ile Asp Val                          405  - #               410  - #               415              - - gat tca gga aac tct tat gat atc tac ata cc - #a tct cat att cag ggc       1296                                                                         Asp Ser Gly Asn Ser Tyr Asp Ile Tyr Ile Pr - #o Ser His Ile Gln Gly                       420      - #           425      - #           430                  - - aat atc act cct cat gct att gtc atc ttg cc - #t aaa                     - #   1332                                                                      Asn Ile Thr Pro His Ala Ile Val Ile Leu Pr - #o Lys                                   435          - #       440                                          __________________________________________________________________________

What is claimed is:
 1. An isolated polytide comprising the amino acidsequence of SEQ ID NO:5.
 2. An isolated polypeptide consisting of theamino acid sequence of SEQ ID NO:5.
 3. The polypeptide of claim 1further comprising heterologous amino acid sequences.
 4. The polypeptideof claim 2 further comprising heterologous amino acid sequences.
 5. Anisolated polypeptide which is encoded by the nucleotide sequence of thehuman DNA insert of the plasmid deposited with ATCC as Accession Number203306.
 6. The polypeptide of claim 5, further comprising heterologousamino acid sequences.